Monoclonal and polyclonal antibodies recognizing coagulase-negative staphylococcal proteins

ABSTRACT

Monoclonal and polyclonal antibodies are provided which recognize and bind to the SdrG protein of  S. epidermidis , and more particularly to antibodies which recognize specific domains of the SdrG protein, namely the SdrG N1N2N3 protein (amino acids 50-597), the SdrG N2N3 protein (amino acids 273-597) and a truncated version of N2N3 identified as SdrG TR2 (amino acids 273-577). The antibodies of the invention, as well as pharmaceutical compositions incorporating these antibodies, are particularly useful in treating or preventing infections caused by coagulase-negative staphylococci

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the benefit of U.S. provisionalapplication Ser. No. 60/361,324, filed Mar. 5, 2002.

FIELD OF THE INVENTION

[0002] The present invention relates to the fields of microbiology,molecular biology, and immunology and more particularly relates to newlyidentified monoclonal antibodies, the use of monoclonal antibodies, aswell as the production of such monoclonal antibodies and recombinanthost cells transformed with the DNA encoding monoclonal antibodies toprevent, treat, or diagnose coagulase-negative staphylococcal infectionsin man and animals. The invention includes murine, chimeric, humanized,and human monoclonal antibodies, as well as fragments, regions andderivatives thereof. In addition, the invention relates to polyclonalantibodies generated against specific domains of the SdrG protein whichare useful in treating or preventing coagulase-negative staphylococcalinfections. The antibodies detailed in this invention have beengenerated from SdrG proteins such as SdrG N1N2N3, N2N3 and TR2, andspecifically recognize SdrG, a fibrinogen binding MSCRAMM® proteinexpressed by coagulase-negative staphylococci such as S. epidermidis.

BACKGROUND OF THE INVENTION

[0003] Coagulase-negative staphylococci, such as Staphylococcusepidermidis, are generally avirulent commensal organisms of the humanskin and the principle etiologic agent of infections of peripheral andcentral venous catheters, prosthetic heart valves, artificial joints,and other prosthetic devices. S. epidermidis bacteremia has anattributable mortality rate of 10-34% and results in an excess hospitalstay of 8 days, with costs for such a stay reaching $6,000.00 or moreper case. Despite its importance as a nosocomial pathogen, relativelylittle is known about the pathogenesis of these infections or thevirulence determinants of this organism. Initial localized infections ofindwelling medical devices can lead to more serious invasive infectionssuch as septicemia, osteomyelitis, and endocarditis. Vascular cathetersare thought to become infected when microorganisms gain access to thedevice, and hence the bloodstream, by migration from the skin surfacedown the transcutaneous portion of the catheter. In infectionsassociated with medical devices, plastic and metal surfaces becomecoated with host plasma and matrix proteins such as fibrinogen,vitronectin and fibronectin shortly after implantation.

[0004] It is now well established that the ability of coagulase-negativestaphylococci to adhere to these proteins is of crucial importance forinitiating infection. Bacterial or microorganism adherence is thought tobe the first crucial step in the pathogenesis of a prosthetic deviceinfection. A number of factors influence an organism's ability to adhereto prosthetic material. These include characteristics of themicroorganism and the biomaterial, and the nature of the ambient milieu.The initial attraction between the organism and the host is influencedby nonspecific forces such as surface charge, polarity, Van der Waalforces and hydrophobic interactions. The critical stage of adherenceinvolves specific interactions between MSCRAMM® proteins and immobilizedhost proteins.

[0005] To date, investigation concerning the adherence of coagulasenegative staphylococci to biomaterials has concerned itself primarilywith the role of the extracellular polysaccharide or glycocalyx, alsoknown as slime. Despite intensive study however, the proposed role ofslime in the pathogenesis of disease or even its composition remaindebated. Drewry. D. T., L Gailbraith. B. I. Wilkinson, and S. G.Wilkinson. 1990. Staphylococcal Slime: A Cautionary Tale, I. Clin.MicrobioL28:1292-1296. Currently, extracellular slime is thought to playa role in the later stages of adherence and persistence of infection. Itmay serve as an ion exchange resin to optimize a local nutritionalenvironment, prevent penetration of antibiotics into the macro-colonyand protect bacteria from phagocytic host defense cells. Peters et alhave shown by electron microscopy studies that extracellularpolysaccharide appears in the later stages of attachment and is notpresent during the initial phase of adherence. O. Peters, R. Locci. andG. Pulverer. 1982. Adherence and Growth of Coagulase-NegativeStaphylococci on Surfaces in Intravenous Catheters. I. Infect. Dis.65146:479-482. Hogt et al demonstrated that removal of the extracellularslime layer by repeated washing does not diminish the ability of S.epidermidis to adhere to biomaterials. Hogt. A. H., I. Dankert, I. A.DeVries. and I. Feijen, 1983. Adhesion of Coagulase-NegativeStaphylococci to Biomaterials. J. Gen. Microbial. 129:2959-2968.

[0006] Thus, the study of the extracellular polysaccharide orexopolysaccharide has lended little to prevention of initial adherenceby the bacteria. Several other studies have identified other potentialadhesins of S. epidermidis including the polysaccharide adhesion (PS/A)observed by Tojo et at. Tojo, M., N. Yamashita, D. A. Goldmann. and G.B. Pier, 1988. Isolation and Characterization of a CapsularPolysaccharide Adhesin 10 from Staphylococcus epidermidis. J. InfectDis. 157:713-722; and the slime associated antigen at (SAA) ofChristensen et al. Christensen. G. D., Barker, L. P., Manhinnes, T. P.,Baddour, L. M., Simpson. W. A. Identification of an Antigenic Marker ofSlime Production for Staphylococcus epidermidis. Infect Immun. 1990;58:2906-2911.

[0007] It has been demonstrated that PS/A is a complex mixture ofmonosaccharides and purified PS/A blocks adherence of PS/A producingstrains of S. epidermidis. In an animal model of endocarditis antibodiesdirected against PS/A was protective. However it is not clear whetherthis protective effect was specific, related to anti-adhesive effects ofthe antibody or due to a more generalized increase in the efficiency ofopsonophagocytosis of blood borne bacteria. It has been hypothesizedthat each functions in different stages of the adherence process withone or more of these adhesins responsible for initial attraction whileother are needed for aggregation in the macro-colonies. Despite all ofthese studies, factors involved in the initial adherence of S.epidermidis to biomaterials remain largely unknown and equally unknownis a practical method for preventing the first stage of infection,adherence.

[0008] Another particular problem in the medical field has been theprevention and/or treatment of coagulase negative staphylococcalinfections in low birth weight infants (LBW) by passive immunizationwith SdrG mAb(s). LBW infants are defined as those infants born between500-1500 g. Premature infants are born before a sufficient transfer ofprotective maternal antibodies through the placenta takes place. Thecombination of insufficient antibodies, blood losses for diagnosticpurposes, less efficient phagocytosis, microbial intestinal overgrowthunder selection pressure from antimicrobial treatment, and repeatedinvasion of otherwise sterile sites by indwelling catheters, are some ofthe reasons for the very high nosocomial infection rates in thisvulnerable population.

[0009] It thus remains a challenge to develop compositions and methodsfor treating and preventing infections by coagulase-negativestaphylococci, and in particular there is a great need to treat orprevent nosocomial infection in vulnerable neonates.

SUMMARY OF THE INVENTION

[0010] Accordingly, it is an object of the present invention to providemonoclonal antibodies capable of recognizing and binding to surfaceproteins such as SdrG from coagulase-negative staphylococci such as S.epidermidis.

[0011] It is further an object of the present invention to developcompositions and methods which can be utilized in the treatment orprevention of nosocomial coagulase negative staphylococcal infections inlow birth weight infants (LBW).

[0012] It is still further an object of the present invention to providemonoclonal antibodies which can recognize the coagulase-negativestaphylococcal SdrG protein and other fibrinogen binding proteins andwhich can thus be used in methods and compositions to treat or preventstaphylococcal infections.

[0013] It is yet another object of the present invention to generateantibodies from the SdrG protein domains such as the N1N2N3 protein, theN2N3 protein, or a truncated version thereof, and to utilize theseantibodies in methods of treating or preventing infection in humans andanimals.

[0014] These and other objects are provided by virtue of the presentinvention which comprises the generation of monoclonal and polyclonalantibodies from the S. epidermidis SdrG protein from the SdrG regionsidentified as N1N2N3 (amino acids 50-597) and N2N3 (amino acids273-597), or a truncated version thereof identified as SdrG TR2 (aminoacids 273-577) which recognize and can bind to the SdrG protein andwhich can thus be used in compositions and method to treat or preventinfections. In addition, the present invention encompasses other uses ofthe antibodies of the invention including the preparation of suitablevaccines, the prevention of infection in medical instruments andprosthetic devices, and the provision of kits used to identify aninfection of coagulase-negative staphylococcus.

[0015] These embodiments and other alternatives and modifications withinthe spirit and scope of the disclosed invention will become readilyapparent to those skilled in the art from reading the presentspecification and/or the references cited herein, all of which areincorporated by reference.

BRIEF DESCRIPTION OF THE DRAWING FIGURES

[0016]FIG. 1 is a graphic representation of a Biacore analysis ofanti-SdrG mAbs in accordance with the invention showing inhibition withSdrG—fibrinogen binding.

[0017]FIG. 2 is a graphic representation of anti-SdrG mAbs in accordancewith the invention showing inhibition of SdrG binding to β-fibrinogenpeptide on the Biacore chip.

[0018]FIG. 3 is a graphic representation of inhibition of humanfibrinogen binding to SdrG as shown by ELISA for monoclonal anti-SdrGantibodies in accordance with the present invention.

[0019]FIG. 4 is a graphic representation of inhibition of humanfibrinogen binding of the protein identified as SEQ ID NO:9 as set forthbelow.

[0020]FIG. 5 is a graphic representation of the results observed in asuckling rat pup challenge model of a coagulase-negative staphylococcal(S. epidermidis) infection.

[0021]FIG. 6 is a graphic representation of the results of a centralvenous catheter (CVC) associated infection model of a coagulase-negativestaphylococcal (S. epidermidis) infection.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] In accordance with the present invention, there are providedantibodies which can bind to the SdrG protein of coagulase-negativebacteria such as S. epidermidis, and which have been shown to protectagainst S. aureus infections. The term “antibodies” as used hereinincludes monoclonal, polyclonal, chimeric, single chain, bispecific,simianized, and humanized or primatized antibodies as well as Fabfragments, such as those fragments which maintain the bindingspecificity of the antibodies to the SdrG protein, including theproducts of a Fab immunoglobulin expression library. Generation of anyof these types of antibodies may be accomplished by suitable means wellknown in the art such as those described below. As explained furtherbelow, these antibodies have been generated from and can recognize andthus bind to the S. epidermidis SdrG regions identified as N1N2N3 (aminoacids 50-597) and N2N3 (amino acids 273-597), as well as a truncatedversion of the N2N3 protein identified as TR2 (amino acids 273-597). Ashas been recently shown, S. epidermidis contains surface proteinsstructurally related to S. aureus MSCRAMM® proteins, as set forth inco-pending patent applications including pending U.S. Ser. No.09/386,962, published as WO 00/12689, incorporated herein by reference.In addition, other information concerning staphylococcal MSCRAMM®proteins is disclosed in U.S. Ser. No. 09/386,960, published as WO00/12132, and U.S. Ser. No. 09/386,959, published as WO 00/12131, allincorporated herein by reference. Additional information regardingMSCRAMM® proteins is disclosed in U.S. Pat. No. 6,288,214, incorporatedherein by reference.

[0023] One of the proteins from S. epidermidis, namely the oneidentified as SdrG (serine-aspartate repeat protein G), such asdisclosed in WO 00/12689, has features typical of Gram-positivebacterial proteins that are anchored to the cell wall. This proteinshows significant amino acid sequence homology to ClfA and ClfB from S.aureus including an 500-amino acid-long A region, a SD dipeptide repeatregion, and has features required for cell wall anchoring, including aLPXTG motif.

[0024] To date, no one has described monoclonal antibodies thatspecifically recognize SdrG, exhibit high affinity (>10⁸ KD), and areprotective in animals models of disease. Accordingly, the presentinvention provides for the first time monoclonal antibodies which canspecifically recognize SdrG, can bind it with high affinity, and whichhas been shown to be protective against Staphylococcal infection.

[0025] In accordance with the present invention, and as describedfurther below, antibodies are generated which recognize the SdrG N1N2N3protein at amino acids 50-597 of the S. epidermidis SdrG protein, theSdrGN2N3 protein (amino acids 273-597) and truncated version TR2 protein(amino acids 273-597), and such antibodies may be used in compositionsand methods of treating or preventing coagulase-negative staphylococcalinfection. In the first aspect of the invention, an isolated and/orpurified version of SdrG N1N2N3, N2N3 and TR2 may be obtained inaccordance with the invention in any suitable manner such as describedbelow. The nucleic acid and amino acid sequences of these proteins areas shown below: SdrG N1N2N3 (50-597): Nucleotide SequenceATGAGAGGATCGCATCACCATCACCATCACGGATCCGAGGAGAATACAGTA (SEQ ID NO:1)CAAGACGTTAAAGATTCGAATATGGATGATGAATTATCAGATAGCAATGATCAGTCCAGTAATGAAGAAAAGAATGATGTAATCAATAATAGTCAGTCAATAAACACCGATGATGATAACCAAATAAAAAAAGAAGAAACGAATAGCAACGATGCCATAGAAAATCGCTCTAAAGATATAACACAGTCAACAACAAATGTAGATGAAAACGAAGCAACATTTTTACAAAAGACCCCTCAAGATAATACTCAGCTTAAAGAAGAAGTGGTAAAAGAACCCTCATCAGTCGAATCCTCAAATTCATCAATGGATACTGCCCAACAACCATCTCATACAACAATAAATAGTGAAGCATCTATTCAAACAAGTGATAATGAAGAAAATTCCCGCGTATCAGATTTTGCTAACTCTAAAATAATAGAGAGTAACACTGAATCCAATAAAGAAGAGAATACTATAGAGCAACCTAACAAAGTAAGAGAAGATTCAATAACAAGTCAACCGTCTAGCTATAAAAATATAGATGAAAAAATTTCAAATCAAGATGAGTTATTAAATTTACCAATAAATGAATATGAAAATAAGGTTAGACCGTTATCTACAACATCTGCCCAACCATCGAGTAAGCGTGTAACCGTAAATCAATTAGCGGCAGAACAAGGTTCGAATGTTAATCATTTAATTAAAGTTACTGATCAAAGTATTACTGAAGGATATGATGATAGTGATGGTATTATTAAAGCACATGATGGTGAAAACTTAATCTATGATGTAACTTTTGAAGTAGATGATAAGGTGAAATCTGGTGATACGATGACAGTGAATATAGATAAGAATACAGTTCCATCAGATTTAACCGATAGTTTTGCAATACCAAAAATAAAAGATAATTCTGGAGAAATCATCGCTACAGGTACTTATGACAACACAAATAAACAAATTACCTACACTTTTACAGATTATGTAGATAAATATGAAAATATTAAAGCGCACCTTAAATTAACATCATACATTGATAAATCAAAGGTTCCAAATAATAACACTAAGTTAGATGTAGAATATAAGACGGCCCTTTCATCAGTAAATAAAACAATTACGGTTGAATATCAAAAACCTAACGAAAATCGGACTGCTAACCTTCAAAGTATGTTCACAAACATAGATACGAAAAACCATACAGTTGAGCAAACGATTTATATTAACCCTCTTCGTTATTCAGCCAAAGAAACAAATGTAAATATTTCAGGGAATGGCGATGAAGGTTCAACAATTATCGAGGATAGTACAATCATTAAAGTTTATAAGGTTGGAGATAATCAAAATTTACCAGATAGTAACAGAATTTATGATTACAGTGAATATGAAGATGTCACAAATGATGAUATGCCCAATTAGGAAATAATAATGACGTGAATATTAATTTTGGTAATATAGATTCACCATATATTATTAAAGTTATTAGTAAATATGACCCTAATAAGGACGATTACAGGACGATACAGCAAACTGTGACAATGCAAACGACTATAAATGAGTATACTGGTGAGTTTAGAACAGCAICCTATGATAATACAATTGCTTTCTCTACAAGTTCAGGTCAAGGACAAGGTGACTTGCCTCCT GAAAAA Amino AcidSequence MRGSHHHHHHGSEENTVQDVKDSNMDDELSDSNDQSSNEEKNDVINNSQSIN (SEQ IDNO:2) TDDDNQIKKEETNSNDAIENRSKDITQSTTNVDENEATFLQKTPQDNTQLKEEVVKEPSSVESSNSSMDTAQQPSHTTINSEASIQTSDNEENSRVSDFANSKIIESNTESNKEENTIEQPNKVREDSITSQPSSYKNIDEKISNQDELLNLPINEYENKVRPLSTTSAQPSSKRVTVNQLAAEQGSNVNHLIKVIDQSITEGYDDSDGIIKAHDAENLIYDVTFEVDDKVKSGDTMTVNIDKNTVPSDLTDSFAIPKIKDNSGEIIATGTYDNTNKQITYTFTDYVDKYENIKAHLKLTSYIDKSKVPNNNTKLDVEYKTALSSVNKTITVEYQKPNENRTANLQSMFTNIDTKNHIVEQTIYINPLRYSAKETNVNISGNGDEGSTIIDDSTIIKVYKVGDNQNLPDSNRIYDYSEYEDVINDDYAQLGNNNDVNINFGNIDSPYIIKVISKYDPNKDDYTTIQQTVTMQTTINEYTGEFRTASYDNTIAFSTSSG QGQGDLPPEKSdrG N2N3 (273-597): Nucleotide Sequence:ATGAGAGGATCGCATCACCATCACCATCACGGATCTCTGGTTCCTAGGGGA (SEQ ID NO:3)TCCGAACAAGGTTCGAATGTTAATCATTTAATTAAAGTTACTGATCAAAGTATTACTGAAGGATATGATGATAGTGATGGTATTATTAAAGCACATGATGCTGAAAACTTAATCTATGATGTAACTTTTGAAGTAGATGATAAGGTGAAATCTGGTGATACGATGACAGTGAATATAGATAAGAATACAGTTCCATCAGATTTAACCGATAGTTTTGCAATACCAAAAATAAAAGATAATTCTGGAGAAATCATCGCTACAGGTACTTATGAGAACACAAATAAACAAATTACCTACACTTTTACAGATTATGTAGATAAATATGAAAATATTAAAGCGCACCTTAAATTAAGATCATACATTGATAAATCAAAGGTTCCAAATAATAACACTAAGTTAGATGTAGAAIATAAGACGGCCCTTTCATCAGTAAATAAAACAATTACGGTTGAATATCAAAAACGTAACGAAAATCGGACTGCTAACCTTCAAAGTATGTTGACAAACATAGATACGAAAAACCATACAGTTGAGCAAACGATTTATATTAACCCTCTTCGTTATTCAGCCAAAGAAACAAATGTAAATATTTCAGGGAATGGCGATGAAGGTTCAACAATTATCGACGATAGTACAATCATTAAAGTTTATAAGGTTGGAGATAATCAAAATTTACCAGATAGTAACAGAATTTATGATTACAGTGAATATGAAGATGTCACAAATGATGATTATGCCCAATTAGGAAATAATAATGACGTGAATATTAATTTTGGTAATATAGATTCACCATATATTATTAAAGTTATTAGTAAATATGACCCTAATAAGGAGGATTACACGACGATACAGCAAACTGTGACAATGCAAACGACTATAAATGAGTATACTGGTGAGTTTAGAACAGCATCCTATGATAATACAATTGCTTTCTCTACAAGTTCAGGTCAAGGACAAGGTGACTTGCCTCCTGAAAAAT Amino Acid SequenceMRGSHHHHHHGSLVPRGSEQGSNVNHLIKVTDQSITEGYDDSDGIIKAHDAENL (SEQ ID NO:4)IYDVTFEVDDKVKSGDTMIVNIDKNTVPSDLTDSFAIPKIKDNSGEIIATGTYDNTNKQITYTFTDYVDKYENIKAHLKLTSYIDKSKVPNNNTKLDVEYKTALSSVNKTITVEYQKPNENRTANLQSMFTNIDTKNHTVEQIIYINPLRYSAKETNVNISGNGDEGSTIIDDSTIIKVYKVGDNQNLPDSNRIYDYSEYEDVINDDYAQLGNNNDVNINFGNIDSPYIIKVISKYDPNKDDYTTIQQTVTMQTTINEYTGEFRTASYDNTIAFSTSS GQGQGDLPPEKSdrG TR2 (273-577): Nucleotide SequenceATGAGAGGATCGCATCACCATCACCATCACGGATCCGAACAAGGTTCGAAT (SEQ ID NO:5)GTTAATCATTTAATTAAAGTTACTGATCAAAGTATTACTGAAGGATATGATGATAGTGATGGTATTATTAAAGCACATGATGCTGAAAACTTAATCTATGATGTAACTTTTGAAGTAGATGATAAGGTGAAATCTGGTGATACGATGACAGTGAATATAGATAAGAATACAGTTCCATCAGATTTAACCGATAGTTTTGCAATACCAAAAATAAAAGATAATTCTGGAGAAATCATCGCTACAGGTACTTATGACAACACAAATAAACAAATTACCTACACTTTTACAGATTATGTAGATAAATATGAAAATATTAAAGCGCACCTTAAATTAACATCATACAHGATAAATCAAAGGTTCCAAATAATAACACTAAGTTAGATGTAGAATATAAGACGGCCCTTTGATCAGTAAATAAAACAATTACGGTTGAATATCAAAAACCTAACGAAAATCGGACTGCTAACCTTCAAAGTATGTTCACAAACATAGATACGAAAAACCATACAGTTGAGCAAACGATTTATATTAACCCTCTTCGTTATTCAGCCAAAGAAACAAATGTAAATATTTCAGGGAATGGCGATGAAGGTTCAACAATTATCGACGATAGTACAATCATTAAAGTTTATAAGGTTGGAGATAATCAAAATTTACCAGATAGTAACAGAATTTATGATTACAGTGAATATGAAGATGTCACAAATGATGATTATGCCCAATTAGGAAATAATAATGACGTGAATATTAATTTTGGTAATATAGATTCACCATATATTATTAAAGTTATTAGTAAATATGACCCTAATAAGGACGATTACACGAGGATACAGCAAACTGTGACAATGCAAACGACTATAAATGAGTATACTGGTGAGTTTAGAACAGCATCC TATTGA Amino AcidSequence MRGSHHHHHHGSEQGSNVNHLIKVTDQSITEGYDDSDGIIKAHDAENLIYDVTF (SEQ IDNO:6) EVDDKVKSGDTMTVNIDKNTVPSDLTDSFAIPKIKDNSGEIIATGTYDNTNKQITYTFTDYVDKYENIKAHLKLTSYIDKSKVPNNNTKLDVEYKTALSSVNKTITVEYQKPNENRTANLQSMFTNIDTKNHTVEQTIYINPLRYSAKETNVNISGNGDEGSTIIDDSTIIKVYKVGDNQNLPDSNRIYDYSEYEDVTNDDYAQLGNNNDVNINFGNIDSPYIIKVISKYDPNKDDYTTIQQTVTMQTTINEYTGEFRTASY

[0026] Accordingly, the present invention encompasses isolated proteinsas described above which have sequences such as SEQ ID NO:2, SEQ ID NO:4or SEQ ID NO:6, as well as isolated proteins encoded by nucleic acidsequences SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5, or degeneratesthereof. In addition, as described further below, the inventionencompasses raising antibodies from these proteins and eliciting aimmune response in humans or animals by administration of an immunogenicamount of the proteins.

[0027] As set forth in more detail below, the monoclonal and polyclonalantibodies of the invention may be prepared in a number of suitable waysthat would be well known in the art. For example, monoclonal antibodiescan be prepared using the well-established Kohler and Milstein methodcommonly used to generate monoclonal antibodies. In one such suitablemethod, mice may be injected intraperitoneally for a prolonged periodwith a purified recombinant protein such as the SdrG N1N2N3 or SdrGN2N3domain or its truncated version TR2 referred to above, followed by atest of blood obtained from the immunized mice to determine reactivityto the purified protein or fragment. Following identification of micereactive to the tested protein, lymphocytes isolated from mouse spleensare fused to mouse myeloma cells to produce hybridomas positive for theantibodies against these proteins which are then isolated and cultured,following by purification and isotyping.

[0028] As described, for example, in J. Biol. Chem. 1999, 274,26939-26945 (incorporated herein by reference), one such suitable meansfor obtaining gene fragments in accordance with the invention, e.g.,those corresponding to the SdrG N1N2N3 protein (aa 50-597), SdrG N2N3protein (aa 273-597) or its truncated version TR2 (aa 273-577) is to usea process wherein they are amplified by using PCR, such as throughsubcloning using E. coli expression vector pQE-30 and transformationusing E. coli strain JM101.

[0029] In a specific example, the proteins of the invention wereobtained in a PCR process wherein SdrGN1N2N3 (representing AA 50-597) orSdrGN2N3 (representing AA 273-597) or its truncated version TR2 (AA273-577) was amplified from S. epidermidis K28 genomic DNA (fromsequences described above) and subcloned into the E. coli expressionvector PQE-30 (Qiagen), which allows for the expression of a recombinantfusion protein containing six histidine residues. This vector wassubsequently transformed into the E. coli strain ATCC 55151, grown in a15-liter fermentor to an optical density (OD₆₀₀) of 0.7 and induced with0.2 mM isopropyl-1-beta-D galactoside (IPTG) for 4 hours. The cells wereharvested using an AG Technologies hollow-fiber assembly (pore size of0.45 μm) and the cell paste frozen at −800 C. Cells were lysed in 1×PBS(10 mL of buffer/1 g of cell paste) using 2 passes through the FrenchPress @ 1100 psi. Lysed cells were spun down at 17,000 rpm for 30minutes to remove cell debris. Supernatant was passed over a 5-mL HiTrapChelating (Pharmacia) column charged with 0.1M NiCl₂. After loading, thecolumn was washed with 5 column volumes of 10 mM Tris, pH 8.0, 100 mMNaCl (Buffer A). Protein was eluted using a 0-100% gradient of 10 mMTris, pH 8.0, 100 mM NaCl 200 mM imidazole (Buffer B) over 30 columnvolumes. SdrGN1N2N3, SdrGN2N3 or TR2 eluted at ˜13% Buffer B (˜26 mMimidazole). Absorbance at 280 nm was monitored. Fractions containingSdrGN1N2N3, SdrGN2N3 or TR2 were dialyzed in 1×PBS.

[0030] The protein was then put through an endotoxin removal protocol.Buffers used during this protocol were made endotoxin free by passingover a 5-mL Mono-Q sepharose (Pharmacia) column. Protein was dividedevenly between 4×15 mL tubes. The volume of each tube was brought to 9mL with Buffer A. 1 mL of 10% Triton X-114 was added to each tube andincubated with rotation for 1 hour at 4° C. Tubes were placed in a 37°C. water bath to separate phases. Tubes were spun down at 2,000 rpm for10 minutes and the upper aqueous phase from each tube was collected andthe detergent extraction repeated. Aqueous phases from the 2ndextraction were combined and passed over a 5-mL IDA chelating (Sigma)column, charged with 0.1M NiCl₂ to remove remaining detergent. Thecolumn was washed with 9 column volumes of Buffer A before the proteinwas eluted with 3 column volumes of Buffer B. The eluant was passed overa 5-mL Detoxigel (Sigma) column and the flow-through collected andreapplied to the column. The flow-through from the second pass wascollected and dialyzed in 1×PBS. The purified product was analyzed forconcentration, purity and endotoxin level before administration into themice.

[0031] As indicated above, generation of the monoclonal antibodies inaccordance with the invention may proceed using any of a number ofconventional methods well known in the art such as the general Kohlerand Milstein technique conventionally used in this field. In onespecific example for preparing the monoclonal antibodies of theinvention, E coli expressed and purified SdrG (N1N2N3, N2N3 or TR2)protein can be used to generate a panel of murine monoclonal antibodies.Briefly, a group of Balb/C or SJL mice received a series of subcutaneousimmunizations of 1-10 mg of protein in solution or mixed with adjuvant.At the time of sacrifice (RIMMS) or seven days after a boost(conventional) serum was collected and titered in ELISA assays againstMSCRAMMs or on whole cells (S. epidermidis). Three days after the finalboost, the spleens or lymph nodes were removed, teased into a singlecell suspension and the lymphocytes harvested. The lymphocytes were thenfused to a P3X63Ag8.653 myeloma cell line (ATCC #CRL-1580). Cell fusion,subsequent plating and feeding were performed according to theProduction of Monoclonal Antibodies protocol from Current Protocols inImmunology (Chapter 2, Unit 2.).

[0032] Any clones that were generated from the fusion were then screenedfor specific anti-SdrG antibody production using a standard ELISA assay.Positive clones were expanded and tested further for activity in a wholebacterial cell binding assay by flow cytometry and SdrGbinding/inhibition of fibrinogen-Clf40 binding by Biacore analysis.Throughout the analysis, the flow rate remained constant at 10 ml/min.Prior to the SdrGN1N2N3, SdrGN2N3 or TR2 injection, test antibody wasadsorbed to the chip via RAM-Fc binding. At time 0, SdrG (N2N3, TR2 orN1N2N3) at a concentration of 30 mg/ml was injected over the chip for 3min followed by 2 minutes of dissociation. This phase of the analysismeasured the relative association and disassociation kinetics of theMab/SdrG interaction. In the second phase of the analysis, the abilityof the Mab bound SdrG to interact and bind fibrinogen was measured.Fibrinogen at a concentration of 100 mg/ml was injected over the chipand after 3 minutes a report point is taken.

[0033] Following the generation of monoclonal antibodies as referred toabove, these antibodies were tested for their ability to bind to wholebacteria. In these tests, bacterial samples (HB, 9142 orSdrG/lactococcus) were collected, washed and incubated with Mab or PBSalone (control) at a concentration of 2 mg/ml after blocking with rabbitIgG (50 mg/ml). Following incubation with antibody, bacterial cells wereincubated with Goat-F_((ab′)2)-Anti-Mouse-F_((ab′)2)-FITC which servedas the detection antibody. After antibody labeling, bacterial cells wereaspirated through the FACScaliber flow cytometer to analyze fluorescenceemission (excitation: 488, emission: 570). For each bacterial strain,10,000 events were collected and measured.

[0034] From these tests, it was shown that SdrG positive hybridomas weregenerated in a frequency of 0.6-10% of the growth positive wells. A fewof the SdrG ELISA positive hybridomas were also positive by Biacoreanalysis and whole cell bacterial binding by flow cytometry. Limitedanalysis demonstrated that Biacore negative, SdrG ELISA positive cloneswere consistently negative in the whole cell binding flow cytometryassay. From this analysis, a very small subpopulation of growth positivehybridoma wells that were SdrG ELISA positive, SdrG Biacore positive andflow cytometry positive on Lactococcus/SdrG were single cell cloned andcharacterized as candidates for potential efficacy against S.epidermidis infection models. These tests showed that monoclonalantibodies generated in accordance with the invention were effective ininhibiting or preventing infection by S. epidermidis and can thus beused in many therapeutic and other useful applications as set forthfurther below.

[0035] In addition to monoclonal antibodies, the present invention alsocontemplates generating polyclonal antibodies from the SdrG proteins asset forth above, as well as other proteins that will generate antibodiesthat can recognize SdrG proteins such as those described herein. Suchpolyclonal antibodies may be generated in any of a number of suitableways well known in the art, such as the introduction of a purified SdrGprotein such as those described herein into a suitable animal host,followed by isolation and purification of the generated antibodiesproduced in the host animal. In general, while it is preferred to useisolated and/or purified recombinant forms of the proteins to generateantibodies in accordance with the invention, antibodies may be generatedas well from natural isolated and/or purified forms of these proteins.

[0036] In accordance with the invention, antibodies are thus producedwhich are generated from SdrG proteins N1N2N3, N2N3, and TR2, and suchantibodies are capable of recognizing and binding SdrG proteins as wellas other fibrinogen binding proteins from S. epidermidis including theproteins described further below. The isolated antibodies and proteinsof the invention can also be utilized in many therapeutic applications,and such applications are described in more detail below.

[0037] Vaccines Humanized Antibodies and Adjuvants

[0038] The isolated antibodies of the present invention, or the isolatedproteins as described above, may also be utilized in the development ofvaccines for active and passive immunization against bacterialinfections, as described further below. Further, when administered aspharmaceutical composition to a wound or used to coat medical devices orpolymeric biomaterials in vitro and in vivo, the antibodies of thepresent invention, may be useful in those cases where there is aprevious infection because of the ability of these antibodies to furtherrestrict and inhibit bacterial binding to collagen and thus limit theextent and spread of the infection.

[0039] In addition, the antibody may be modified as necessary so that,in certain instances, it is less immunogenic in the patient to whom itis administered. For example, if the patient is a human, the antibodymay be “humanized” by transplanting the complimentarity determiningregions of the hybridoma-derived antibody into a human monoclonalantibody as described, e.g., by Jones et al., Nature 321:522-525 (1986)or Tempest et al. Biotechnology 9:266-273 (1991) or “veneered” bychanging the surface exposed murine framework residues in theimmunoglobulin variable regions to mimic a homologous human frameworkcounterpart as described, e.g., by Padlan, Molecular 1 mm. 28:489-498(1991), these references incorporated herein by reference. Even further,when so desired, the monoclonal antibodies of the present invention maybe administered in conjunction with a suitable antibiotic to furtherenhance the ability of the present compositions to fight bacterialinfections.

[0040] In a preferred embodiment, the antibodies may also be used as apassive vaccine which will be useful in providing suitable antibodies totreat or prevent a bacterial infection. As would be recognized by oneskilled in this art, a vaccine may be packaged for administration in anumber of suitable ways, such as by parenteral (i.e., intramuscular,intradermal or subcutaneous) administration or nasopharyngeal (i.e.,intranasal) administration. One such mode is where the vaccine isinjected intramuscularly, e.g., into the deltoid muscle, however, theparticular mode of administration will depend on the nature of thebacterial infection to be dealt with and the condition of the patient.The vaccine is preferably combined with a pharmaceutically acceptablecarrier to facilitate administration, and the carrier is usually wateror a buffered saline, with or without a preservative. The vaccine may belyophilized for resuspension at the time of administration or insolution.

[0041] The preferred dose for administration of an antibody compositionin accordance with the present invention is that amount will beeffective in preventing of treating a bacterial infection, and one wouldreadily recognize that this amount will vary greatly depending on thenature of the infection and the condition of a patient. An “effectiveamount” of antibody or pharmaceutical agent to be used in accordancewith the invention is intended to mean a nontoxic but sufficient amountof the agent, such that the desired prophylactic or therapeutic effectis produced. Accordingly, the exact amount of the antibody or aparticular agent that is required will vary from subject to subject,depending on the species, age, and general condition of the subject, theseverity of the condition being treated, the particular carrier oradjuvant being used and its mode of administration, and the like.Accordingly, the “effective amount” of any particular antibodycomposition will vary based on the particular circumstances, and anappropriate effective amount may be determined in each case ofapplication by one of ordinary skill in the art using only routineexperimentation. The dose should be adjusted to suit the individual towhom the composition is administered and will vary with age, weight andmetabolism of the individual. The compositions may additionally containstabilizers or pharmaceutically acceptable preservatives, such asthimerosal (ethyl(2-mercaptobenzoate-S)mercury sodium salt) (SigmaChemical Company, St. Louis, Mo.).

[0042] In addition, an active vaccine in accordance with the inventionis provided wherein an immunogenic amount of an isolated protein asdescribed above is administered to a human or animal patient in need ofsuch a vaccine. The vaccine may also comprise a suitable,pharmaceutically acceptable vehicle, excipient or carrier such asdescribed above. As indicated above, an “immunogenic amount” of theantigen to be used in accordance with the invention is intended to meana nontoxic but sufficient amount of the agent, such that an immunogenicresponse will be elicited in the host so that the desired prophylacticor therapeutic effect is produced. Accordingly, the exact amount of theantigen that is required will vary from subject to subject, depending onthe species, age, and general condition of the subject, the severity ofthe condition being treated, the particular carrier or adjuvant beingused and its mode of administration, and the like. Similarly, the“immunogenic amount” of any such antigenic vaccine composition will varybased on the particular circumstances, and an appropriate immunogenicamount may be determined in each case of application by one of ordinaryskill in the art using only routine experimentation. The dose should beadjusted to suit the individual to whom the composition is administeredand will vary with age, weight and metabolism of the individual.

[0043] In addition, the antibody compositions of the present inventionand the vaccines as described above may also be administered with asuitable adjuvant in an amount effective to enhance the immunogenicresponse against the conjugate. For example, suitable adjuvants mayinclude alum (aluminum phosphate or aluminum hydroxide), which is usedwidely in humans, and other adjuvants such as saponin and its purifiedcomponent Quil A, Freund's complete adjuvant, and other adjuvants usedin research and veterinary applications. Still other chemically definedpreparations such as muramyl dipeptide, monophosphoryl lipid A,phospholipid conjugates such as those described by Goodman-Snitkoff etal. J. Immunol 147:410-415 (1991) and incorporated by reference herein,encapsulation of the conjugate within a proteoliposome as described byMiller et al., J. Exp. Med. 176:1739-1744 (1992) and incorporated byreference herein, and encapsulation of the protein in lipid vesiclessuch as Novasome lipid vesicles (Micro Vescular Systems, Inc., Nashua,N.H.) may also be useful.

[0044] Pharmaceutical Compositions

[0045] As would be recognized by one skilled in the art, the antibodiesof the present invention may also be formed into suitable pharmaceuticalcompositions for administration to a human or animal patient in order totreat or prevent an infection caused by coagulase-negativestaphylococcal bacteria. Pharmaceutical compositions containing theantibodies of the present invention as defined and described above maybe formulated in combination with any suitable pharmaceutical vehicle,excipient or carrier that would commonly be used in this art, includingsuch as saline, dextrose, water, glycerol, ethanol, other therapeuticcompounds, and combinations thereof. As one skilled in this art wouldrecognize, the particular vehicle, excipient or carrier used will varydepending on the patient and the patient's condition, and a variety ofmodes of administration would be suitable for the compositions of theinvention, as would be recognized by one of ordinary skill in this art.Suitable methods of administration of any pharmaceutical compositiondisclosed in this application include, but are not limited to, topical,oral, anal, vaginal, intravenous, intraperitoneal, intramuscular,subcutaneous, intranasal and intradermal administration.

[0046] For topical administration, the composition is formulated in theform of an ointment, cream, gel, lotion, drops (such as eye drops andear drops), or solution (such as mouthwash). Wound or surgicaldressings, sutures and aerosols may be impregnated with the composition.The composition may contain conventional additives, such aspreservatives, solvents to promote penetration, and emollients. Topicalformulations may also contain conventional carriers such as cream orointment bases, ethanol, or oleyl alcohol.

[0047] Additional forms of antibody compositions, and other informationconcerning compositions, methods and applications with regard to otherMSCRAMM® proteins and MSCRAMM® peptides will generally also beapplicable to the present invention involving monoclonal antibodies andare disclosed, for example, in U.S. Pat. No. 6,288,214 (Hook et al.),incorporated herein by reference.

[0048] The antibody compositions of the present invention which aregenerated in particular against the SdrG proteins as set forth above mayalso be administered with a suitable adjuvant in an amount effective toenhance the immunogenic response against the conjugate. For example,suitable adjuvants may include alum (aluminum phosphate or aluminumhydroxide), which is used widely in humans, and other adjuvants such assaponin and its purified component Quil A, Freund's complete adjuvant,RIBI adjuvant, and other adjuvants used in research and veterinaryapplications. Still other chemically defined preparations such asmuramyl dipeptide, monophosphoryl lipid A, phospholipid conjugates suchas those described by Goodman-Snitkoff et al. J. Immunol. 147:410-415(1991) and incorporated by reference herein, encapsulation of theconjugate within a proteoliposome as described by Miller et al., J. Exp.Med. 176:1739-1744 (1992) and incorporated by reference herein, andencapsulation of the protein in lipid vesicles such as Novasome™ lipidvesicles (Micro Vescular Systems, Inc., Nashua, N.H.) may also beuseful.

[0049] In any event, the antibody compositions of the present inventionwill thus be useful for interfering with, modulating, inhibiting bindinginteractions involving fibrinogen binding proteins as would take placewith bacteria from coagulase-negative staphylococci. Accordingly, thepresent invention will have particular applicability in developingcompositions and methods of preventing or treating coagulase-negativestaphylococcal infection, and in inhibiting binding of staphylococcalbacteria to host tissue and/or cells.

[0050] Methods:

[0051] Treating or Protecting Against Infections

[0052] In accordance with the present invention, methods are providedfor preventing or treating a coagulase-negative staphylococcal infectionwhich comprise administering an effective amount of the antibodies asdescribed above to a human or animal patient in need of such treatmentin amounts effective to treat or prevent the infection. In addition,antibodies in accordance with the invention will be particularly usefulin impairing the binding of a variety of bacteria to fibrinogen, andhave thus proved effective in treating or preventing infection frombacteria such as coagulase-negative staphylococci by inhibiting saidbinding.

[0053] Accordingly, in accordance with the invention, administration ofan effective amount of the antibodies of the present invention in any ofthe conventional ways described above (e.g., topical, parenteral,intramuscular, etc.), and will thus provide an extremely useful methodof treating or preventing coagulase-negative staphylococcal infectionsin human or animal patients. As indicated above, by effective amount ismeant that level of use, such as of an antibody titer, that will besufficient to either prevent adherence of the bacteria, to inhibitbinding of bacteria to host cells and thus be useful in the treatment orprevention of a bacterial infection. As would be recognized by one ofordinary skill in this art, the level of antibody titer needed to beeffective in treating or preventing infections will vary depending onthe nature and condition of the patient, and/or the severity of thepre-existing infection.

[0054] Eliciting an Immune Response

[0055] In accordance with the present invention, a method is providedfor eliciting an immunogenic reaction in a human or animal comprisingadministering to the human or animal an immunologically effective amountof an isolated protein as described above, such as SdrG N1N2N3, SdrGN2N3 or SdrG TR2. As indicated above, an “immunogenic amount” of theantigen to be used in accordance with the invention to obtain animmunogenic reaction is intended to mean a nontoxic but sufficientamount of the agent, such that an immunogenic response will be elicitedin the host so that the desired prophylactic or therapeutic effect isproduced. Accordingly, the exact amount of the isolated protein that isrequired to elicit such a response will vary from subject to subject,depending on the species, age, and general condition of the subject, theseverity of the condition being treated, the particular carrier oradjuvant being used and its mode of administration, and the like. Theinvention also contemplates methods of generating antibodies whichrecognize the SdrG proteins as described above, and suitable methods ofgenerating monoclonal and polyclonal antibodies are described in moredetail above.

[0056] Coating devices

[0057] In accordance with the invention, the antibodies and compositionsas described above may also be utilized to treat or protect againstoutbreaks of coagulase-staphylococcal infections on medical devices andother implanted materials such as prosthetic devices. Medical devices orpolymeric biomaterials that may be advantageously coated with theantibodies and/or compositions described herein include, but are notlimited to, staples, sutures, replacement heart valves, cardiac assistdevices, hard and soft contact lenses, intraocular lens implants(anterior chamber or posterior chamber), other implants such as cornealinlays, kerato-prostheses, vascular stents, epikeratophalia devices,glaucoma shunts, retinal staples, scleral buckles, dental prostheses,thyroplastic devices, laryngoplastic devices, vascular grafts, soft andhard tissue prostheses including, but not limited to, pumps, electricaldevices including stimulators and recorders, auditory prostheses,pacemakers, artificial larynx, dental implants, mammary implants, penileimplants, cranio/facial tendons, artificial joints, tendons, ligaments,menisci, and disks, artificial bones, artificial organs includingartificial pancreas, artificial hearts, artificial limbs, and heartvalves; stents, wires, guide wires, intravenous and central venouscatheters, laser and balloon angioplasty devices, vascular and heartdevices (tubes, catheters, balloons), ventricular assists, blooddialysis components, blood oxygenators, urethral/ureteral/urinarydevices (Foley catheters, stents, tubes and balloons), airway catheters(endotracheal and tracheostomy tubes and cuffs), enteral feeding tubes(including nasogastric, intragastric and jejunal tubes), wound drainagetubes, tubes used to drain the body cavities such as the pleural,peritoneal, cranial, and pericardial cavities, blood bags, test tubes,blood collection tubes, vacutainers, syringes, needles, pipettes,pipette tips, and blood tubing.

[0058] It will be understood by those skilled in the art that the term“coated” or “coating”, as used herein, means to apply the antibody orcomposition as defined above to a surface of the device, preferably anouter surface that would be exposed to a bacterial infection. Thesurface of the device need not be entirely covered by the protein,antibody or active fragment.

[0059] As indicated above, the antibodies of the present invention, oractive portions or fragments thereof, are particularly useful forinterfering with the initial physical interaction between a bacterialpathogen responsible for infection and a mammalian host, such as theadhesion of the bacteria to mammalian extracellular matrix proteins suchas fibrinogen, and this interference with the physical interaction maybe useful both in treating patients and in preventing or reducingbacteria infection on in-dwelling medical devices to make them safer foruse.

[0060] Kits

[0061] In accordance with the present invention, the antibodies of theinvention as set forth above may be used in kits to diagnose aninfection by coagulase-negative staphylococci such as S. epidermidis.Such diagnostic kits are well known in the art and will generally beprepared so as to be suitable for determining the presence of bacteriaor proteins that will bind to the antibodies of the invention. Thesediagnostic kits will generally include the antibodies of the inventionalong with suitable means for detecting binding by that antibody such aswould be readily understood by one skilled in this art. For example, themeans for detecting binding of the antibody may comprise a detectablelabel that is linked to said antibody. These kits can then be used indiagnostic methods to detect the presence of a coagulase-negativestaphylococcal infection wherein one obtains a sample suspected of beinginfected by one or more coagulase-negative staphylococcal bacteria, suchas a sample taken from an individual, for example, from one's blood,saliva, tissues, bone, muscle, cartilage, or skin, introduces to thesample one or more of the antibodies as set forth herein, and thendetermines if the antibodies bind to the sample which would indicatedthe presence of such bacteria in the sample.

[0062] In short, the antibodies of the present invention as describedabove can be extremely useful in inhibiting fibrinogen binding and intreating or preventing the infection of humans, animals, or medicaldevices and prosthesis that can be caused by coagulase-negativestaphylococcal bacteria. In particular, the present invention will be ofimportance in the treatment or prevention of nosocomial coagulasenegative staphylococcal infections in low birth weight infants (LBW).

EXAMPLES

[0063] The following examples are provided which exemplify aspects ofthe preferred embodiments of the present invention. It should beappreciated by those of skill in the art that the techniques disclosedin the examples which follow represent techniques discovered by theinventors to function well in the practice of the invention, and thuscan be considered to constitute preferred modes for its practice.However, those of skill in the art should, in light of the presentdisclosure, appreciate that many changes can be made in the specificembodiments which are disclosed and still obtain a like or similarresult without departing from the spirit and scope of the invention.

Example 1 Expression and Purification of SdrG Proteins

[0064] In accordance with the present invention, proteins obtained fromthe relevant domains of the SdrG protein were cloned, expressedrecombinantly and isolated and/or purified. The SdrG N1N2N3 protein(50-597) represents the putative A domain of the SdrG gene. SdrG N2N3protein (273-597) represents the sub-domain required for humanfibrinogen binding. SdrG TR2 protein (273-577) represents the sub-domainrequired for human fibrinogen binding with the C-terminal portionremoved that stabilizes fibrinogen binding. The nucleotide and aminoacid sequences for these proteins are set forth below: 16/30 SdrG N1N2N3(50-597): Nucleotide SequenceATGAGAGGATCGCATCACCATCACCATCACGGATCCGAGGAGAATACAGTA (SEQ ID NO:1)CAAGACGTTAAAGATTCGAATATGGATGATGAATTATCAGATAGCAATGATCAGTCCAGTAATGAAGAAAAGAATGATGTAATCAATAATAGTCAGTCAATAAACACCGATGATGATAACCAAATAAAAAAAGAAGAAACGAATAGCAACGATGCCATAGAAAATCGCTCTAAAGATATAACACAGTCAACAACAAATGTAGATGAAAACGAAGCAACATTTTTACAAAAGACCCCTCAAGATAATACTCAGCTTAAAGAAGAAGTGGTAAAAGAACCCTCATCAGTCGAATCCTCAAATTCATCAATGGATACTGCCCAACAACCATCTCATACAACAATAAATAGTGAAGCATCTATTCAAACAAGTGATAATGAAGAAAATTCCCGCGTATCAGATTTTGCTAACTCTAAAATAATAGAGAGTAACACTGAATCCAATAAAGAAGAGAATACTATAGAGCAACCTAACAAAGTAAGAGAAGATTCAATAACAAGTCAACCGTCTAGCTATAAAAATATAGATGAAAAAATTTCAAATCAAGATGAGTTATTAAATTTACCAATAAATGAATATGAAAATAAGGTTAGACCGHATCTACAACATCTGCCCAACCATCGAGTAAGCGTGTAACCGTAAATCAATTAGCGGCAGAACAAGGTTCGAATGTTAATCATTTAATTAAAGTTACTGATCAAAGTATTACTGAAGGATATGATGATAGTGATGGTATTATTAAAGCACATGATGCTGAAAACTTAATCTATGATGTAACTTTTGAAGTAGATGATAAGGTGAAATCTGGTGATACGATGACAGTGAATATAGATAAGAATACAGTTCCATCAGATTTAACCGATAGTTTTGCAATACCAAAAATAAAAGATAATTCTGGAGAAATCATCGCTACAGGTACTTATGACAACACAAATAAACAAATTACCTACACTTTTACAGATTATGTAGATAAATATGAAAATATTAAAGCGCACCTTAAATTAACATCATACATTGATAAATCAAAGGTTCCAAATAATAACACTAAGTIAGATGTAGAATATAAGACGGCCCTTTCATCAGTAAATAAAACAATTACGGTTGAATATCAAAAACCTAACGAAAATCGGACTGCTAACCTTCAAAGTATGTTCACAAACATAGATACGAAAAACCATACAGTTGAGCAAACGATTTATATTAACCCTCTTCGTTATTCAGCCAAAGAAACAAATGTAAATATTTCAGGGAATGGCGATGAAGGTTCAACAATTATCGAGGATAGTACAATCATTAAAGTTTATAAGGTTGGAGATAATCAAAATTTACCAGATAGTAACAGAATTTATGATTACAGTGAATATGAAGATGTCACAAATGATGATTATGCCCAATTAGGAAATAATAATGACGTGAATATTAATTTTGGTAATATAGATTCACCATATATTATTAAAGTTATTAGTAAATATGACCCTAATAAGGACGATTACACGACGATACAGCAAACTGTGACAATGCAAACGACTATAAATGAGTATACTGGTGAGTTTAGAACAGCATCCTATGATAATACAATTGCTTTCTCTACAAGTTCAGGTCAAGGACAAGGTGACTTGCCTGCT GAAAAA Amino AcidSequence MRGSHHHHHHGSEENTVQDVKDSNMDDELSDSNDQSSNEEKNDVINNSQSIN (SEQ IDNO:2) TDDDNQIKKEETNSNDAIENRSKDITQSTTNVDENEAIFLQKIPQDNTQLKEEVVKEPSSVESSNSSMDTAQQPSHTTINSEASIQTSDNEENSRVSDFANSKIIESNTESNKEENTIEQPNKVREDSITSQPSSYKNIDEKISNQDELLNLPINEYENKVRPLSTTSAQPSSKRVTVNQLAAEQGSNVNHLIKVTDQSITEGYDDSDGIIKAHDAENLIYDVTFEVDDKVKSGDTMTVNIDKNTVPSDLTDSFAIPKIKDNSGEIIATGTYDNTNKQITYTFIDYVDKYENIKAHLKLTSYIDKSKVPNNNTKLDVEYKTALSSVNKTITVEYQKPNENRTANLQSMFTNIDTKNHTVEQTIYINPLRYSAKETNVNISGNGDEGSTIIDDSTIIKVYKVGDNQNLPDSNRIYDYSEYEDVTNDDYAQLGNNNDVNINFGNIDSPYIIKVISKYDPNKDDYTTIQQTVTMQTTINEYTGEFRTASYDNTIAFSTSSG QGQGDLPPEKSdrG N2N3 (273-597): Nucleotide SequenceATGAGAGGATCGCATCACCATCACCATCACGGATCTCTGGTTCCTAGGGGA (SEQ ID NO:3)TCCGAACAAGGTTCGAATGTTAATCATTTAATTAAAGTTACTGATCAAAGTATTACTGAAGGATATGATGATAGTGATGGTATTATTAAAGCACATGATGCTGAAAACTTAATCTATGATGTAACTTTTGAAGTAGATGATAAGGTGAAATCTGGTGATACGATGACAGTGAATATAGATAAGAATACAGTTCCATCAGATTTAACCGATAGTTTTGCAATACCAAAAATAAAAGATAATTCTGGAGAAATCATCGCTACAGGTACTTATGACAACACAAATAAACAAATTACCTACACTTTTACAGATTATGTAGATAAATATGAAAATATTAAAGCGCACCTTAAATTAACATCATACATTGATAAATCAAAGGTTCCAAATAATAACACTAAGTTAGATGTAGAATATAAGACGGCCCTTTCATCAGTAAATAAAACAATTACGGTTGAATATCAAAAACCTAACGAAAATCGGACTGCTAACCTTCAAAGTATGTTCACAAACATAGATACGAAAAACCATACAGTTGAGCAAACGATTTATATTAACCCTCTTCGTTATTCAGCCAAAGAAACAAATGTAAATATTTCAGGGAATGGGGATGAAGGTTCAACAATTATCGACGATAGTACAATCATTAAAGTTTATAAGGTTGGAGATAATCAAAATTTACCAGATAGTAACAGAATTTATGATTACAGTGAATATGAAGATGTCACAAATGATGATTATGCCCAATTAGGAAATAATAATGACGTGAATATTAATTTTGGTAATATAGATTCACCATATATTATTAAAGTTATTAGTAAATATGACCCTAATAAGGACGATTACACGACGATACAGCAAACTGTGACAATGCAAACGACTATAAATGAGTATACTGGTGAGTTTAGAACAGCATCCTATGATAATACAATTGCTTTCTCTACAAGTTCAGGTCAAGGACAAGGTGACTTGCCTCCTGAAAAAT Amino Acid SequenceMRGSHHHHHHGSLVPRGSEQGSNVNHLIKVIDQSITEGYDDSDGIIKAHDAENL (SEQ ID NO:4)IYDVTFEVDDKVKSGDTMTVNIDKNTVPSDLTDSFAIPKIKDNSGEIIATGTYDNTNKQITYTFTDYVDKYENIKAHLKLTSYIDKSKVPNNNTKLDVEYKTALSSVNKTITVEYQKPNENRTANLQSMFTNIDTKNHTVEQTIYINPLRYSAKETNVNISGNGDEGSTIIDDSTIIKVYKVGDNQNLPDSNRIYDYSEYEDVTNDDYAQLGNNNDVNINFGNIDSPYIIKVISKYDPNKDDYTTIQQTVTMQTTINEYTGEFRTASYDNTIAFSTSS GQGQGDLPPEKSdrG TR2 (273-577): Nucleotide SequenceATGAGAGGATCGCATCACCATCACCATCACGGATCCGAACAAGGTTCGAAT (SEQ ID NO:5)GTTAATCAT1TAATTAAAGTTACTGATCAAAGTATTACTGAAGGATATGATGATAGTGATGGTATTATTAAAGCACATGATGCTGAAAACTTAATCTATGATGTAACTTTTGAAGTAGATGATAAGGTGAAATCTGGTGATACGATGACAGTGAATATAGATAAGAATACAGTTCCATCAGATTTAACCGATAGTTTTGCAATACCAAAAATAAAAGATAATTCTGGAGAAATCATCGCTACAGGTACTTATGACAACACAAATAAACAAATTACCTACACTTTTACAGATTATGTAGATAAATATGAAAATATTAAAGCGCACCTTAAATTAACATCATACATTGATAAATCAAAGGTTCCAAATAATAACACTAAGTTAGATGTAGAATATAAGACGGCCTTTCATCAGTAAATAAAACAATTACGGTTGAATATCAAAAACCTAACGAAAATCGGACTGCTAACCTTCAAAGTATGTTCACAAACATAGATACGAAAAACCATACAGTTGAGCAAACGATTTATATTAACCCTCTTCGTTATTCAGCCAAAGAAACAAATGTAAATATTTCAGGGAATGGCGATGAAGGTTCAACAATTATCGAGGATAGTACAATCATTAAAGTTTATAAGGTTGGAGATAATCAAAATTTACCAGATAGTAACAGAATTTATGATTACAGTGAATATGAAGATGTCACAAATGATGATTATGCCCAATTAGGAAATAATAATGACGTGAATATTAATTTTGGTAATATAGATTCACCATATATTATTAAAGTTATTAGTAAATATGACCCTAATAAGGACGATTACACGACGATACAGCAAACTGTGACAATGCAAACGACTAIAAATGAGTATACTGGTGAGTTTAGAACAGCATCC TATTGA Amino AcidSequence MRGSHHHHHHGSEQGSNVNHLIKVIDQSITEGYDDSDGIIKAHDAENLIYDVTF (SEQ IDNO:6) EVDDKVKSGDTMTVNIDKNTVPSDLTDSFAIPKIKDNSGEIIATGTYDNTNKQITYTFTDYVDKYENIKAHLKLTSYIDKSKVPNNNTKLDVEYKTALSSVNKTITVEYQKPNENRTANLQSMFTNIDTKNHTVEQTIYINPLRYSAKETNVNISGNGDEGSTIIDDSTIIKVYKVGDNQNLPDSNRIYDYSEYEDVTNDDYAQLGNNNDVNINFGNIDSPYIIKVISKYDPNKDDYTTIQQTVTMQTTINEYTGEFRTASY

[0065] Protein Production and Purification

[0066] Using PCR, SdrGN1N2N3 (representing AA 50-597) or its subdomainssuch as SdrGN2N3 (representing AA 273-597) or its truncate TR2 (AA273-577) were amplified from S. epidermidis K28 genomic DNA (fromsequences described above) and subcloned into the E. coli expressionvector PQE-30 (Qiagen), which allows for the expression of a recombinantfusion protein containing six histidine residues. This vector wassubsequently transformed into the E. coli strain ATCC 55151, grown in a15-liter fermentor to an optical density (OD₆₀₀) of 0.7 and induced with0.2 mM isopropyl-1-beta-D galactoside (IPTG) for 4 hours. The cells wereharvested using an AG Technologies hollow-fiber assembly (pore size of0.45 □m) and the cell paste frozen at −80° C. Cells were lysed in 1×PBS(10 mL of buffer/1 g of cell paste) using 2 passes through the FrenchPress @ 1100 psi. Lysed cells were spun down at 17,000 rpm for 30minutes to remove cell debris. Supernatant was passed over a 5-mL HiTrapChelating (Pharmacia) column charged with 0.1M NiCl₂. After loading, thecolumn was washed with 5 column volumes of 10 mM Tris, pH 8.0, 100 mMNaCl (Buffer A). Protein was eluted using a 0-100% gradient of 10 mMTris, pH 8.0, 100 mM NaCl, 200 mM imidazole (Buffer B) over 30 columnvolumes. SdrGN1N2N3, SdrGN2N3 or TR2 eluted at ˜13% Buffer B (˜26 mMimidazole). Absorbance at 280 nm was monitored. Fractions containingSdrGN1N2N3, SdrGN2N3 or TR2 were dialyzed in 1×PBS.

[0067] The protein was then put through an endotoxin removal protocol.Buffers used during this protocol were made endotoxin free by passingover a 5-mL Mono-Q sepharose (Pharmacia) column. Protein was dividedevenly between 4×15 mL tubes. The volume of each tube was brought to 9mL with Buffer A. 1 mL of 10% Triton X-114 was added to each tube andincubated with rotation for 1 hour at 4° C. Tubes were placed in a 37°C. water bath to separate phases. Tubes were spun down, at 2,000 rpm for10 minutes and the upper aqueous phase from each tube was collected andthe detergent extraction repeated. Aqueous phases from the 2ndextraction were combined and passed over a 5-mL IDA chelating (Sigma)column, charged with 0.1M NiCl₂ to remove remaining detergent. Thecolumn was washed with 9 column volumes of Buffer A before the proteinwas eluted with 3 column volumes of Buffer B. The eluant was passed overa 5-mL Detoxigel (Sigma) column and the flow-through collected andreapplied to the column. The flow-through from the second pass wascollected and dialyzed in lx PBS. The purified product was analyzed forconcentration, purity and endotoxin level before administration into themice.

Example 2 Immunization Strategies for Monoclonal Antibody Production

[0068] With the goal of generating and characterizing monoclonalantibodies (mAbs), strategies were formulated to generate mAbs againstSdrG that were of high affinity, able to interrupt or restrict thebinding of fibrinogen to SdrG and demonstrate therapeutic efficacy invivo. E. coli expressed and purified SdrG (N1N2N3, N2N3 or TR2) proteinwas used to generate a panel of murine monoclonal antibodies. Briefly, agroup of Balb/C or SJL mice received a series of subcutaneousimmunizations of 1-10 mg of protein in solution or mixed with adjuvantas described below in Table I: TABLE I Immunization Schemes Day Amount(μg) Route Adjuvant RIMMS Injection #1 0 5 Subcutaneous FCA/RIBI #2 2 1Subcutaneous FCA/RIBI #3 4 1 Subcutaneous FCA/RIBI #4 7 1 SubcutaneousFCA/RIBI #5 9 1 Subcutaneous FCA/RIBI Conventional Injection Primary 0 5Subcutaneous FCA Boost #1 14 1 Intraperitoneal RIBI Boost #2 28 1Intraperitoneal RIBI Boost #3 42 1 Intraperitoneal RIBI

[0069] At the time of sacrifice (RIMMS) or seven days after a boost(conventional) serum was collected and titered in ELISA assays againstMSCRAMMs or on whole cells (S. epidermidis). Three days after the finalboost, the spleens or lymph nodes were removed, teased into a singlecell suspension and the lymphocytes harvested. The lymphocytes were thenfused to a P3X63Ag8.653 myeloma cell line (ATCC #CRL-1580). Cell fusion,subsequent plating and feeding were performed according to theProduction of Monoclonal Antibodies protocol from Current Protocols inImmunology (Chapter 2, Unit 2.).

Example 3 Screening and Selection of Anti-SdrG Monoclonal Antibodies

[0070] Any clones that were generated from the fusion were then screenedfor specific anti-SdrG antibody production using a standard ELISA assay.Positive clones were expanded and tested further for activity in a wholebacterial cell binding assay by flow cytometry and SdrG binding byBiacore analysis.

[0071] ELISA Analysis

[0072] Immulon 2-HB high-binding 96-well microtiter plates (Dynex) werecoated with 1 μg/well of rClfA-(40-559) in 1×PBS, pH 7.4 and incubatedfor 2 hours at room temperature. All washing steps in ELISAs wereperformed three times with 1×PBS, 0.05% Tween-20 wash buffer. Plateswere washed and blocked with a 1% BSA solution at room temperature for 1hour before hybridoma supernatant samples were added to wells. Plateswere incubated with samples and relevant controls such as media alonefor one hour at room temperature, washed, and goat anti-mouse IgG-AP(Sigma) diluted 1:5000 in 1×PBS, 0.05% Tween-20, 0.1% BSA was used as asecondary reagent. Plates were developed by addition of 1 mg/ml solutionof 4-nitrophenyl phosphate (pNPP) (Sigma), followed by incubation at 37°C. for 30 minutes. Absorbance was read at 405 nm using a SpectraMax 190Plate Reader (Molecular Devices Corp.). Antibody supernatants that hadan OD₄₀₅≧3 times above background (media alone, ˜0.1 OD) were consideredpositive.

[0073] Biacore Analysis

[0074] Throughout the analysis, the flow rate remained constant at 10ml/min. Prior to the SdrGN1N2N3 or SdrGN2N3/TR2 injection, test antibodywas adsorbed to the chip via RAM-Fc binding. At time 0, SdrG (N2N3, TR2or N1N2N3) at a concentration of 30 mg/ml was injected over the chip for3 min followed by 2 minutes of dissociation. This phase of the analysismeasured the relative association and disassociation kinetics of theMab/SdrG interaction.

[0075] Binding to Whole Bacteria

[0076] Bacterial samples (HB, 9142 or SdrG/lactococcus) were collected,washed and incubated with Mab or PBS alone (control) at a concentrationof 2 mg/ml after blocking with rabbit IgG (50 mg/ml). Followingincubation with antibody, bacterial cells were incubated withGoat-F(ab′)₂-Anti-Mouse-F(ab′)₂-FITC which served as the detectionantibody. After antibody labeling, bacterial cells were aspiratedthrough the FACScaliber flow cytometer to analyze fluorescence emission(excitation: 488, emission: 570). For each bacterial strain, 10,000events were collected and measured. TABLE II SdrG Screening Summary #SdrG # Whole Cell # Growth Positives # Biacore Binding PositivesImmunization Positive by ELISA Positives by Flow (% of Fusion # ProtocolAntigen Wells (% of total) (% of total) total) Fusion 41 RIMMSSdrGN1N2N3 261 26 (10%) 14 (5.4%) 4 (1.5%) Fusion 42 RIMMS SdrGN1N2N3207 8 (3.9%) 4 (1.9%) 0 Fusion 58 RIMMS SdrGN1N2N3 167 6 (3.4%) 6 (3.4%)5 (3%)   Fusion 59 RIMMS SdrGN2N3 164 1 (0.6%) 1 (0.6%) 0 Fusion 62Conventional SdrGN2N3 1440 144 (10%) 74 (5.1%) 19 (1.3%)  Fusion 63Conventional SdrGN2N3 1440 22 (1.5%) 9 (0.6%) 7 (0.5%) Fusion 64Conventional SdrGN1N2N3 2000 32 (1.6%) 8 (0.4%) 7 (0.4%) Fusion 80Conventional SdrGN2N3 1920 52 (2.7%) 11 (0.6%) ND Fusion 81 ConventionalSdrGN2N3 1920 32 (1.8%) 5 (0.3%) ND Fusion 82 Conventional SdrGTR2 14407 (0.5%) 2 (0.1%) ND Fusion 83 Conventional SdrGTR2 1440 21 (1.5%) 14(1%) ND

[0077] From the above analysis, SdrG positive hybridomas were generatedin a frequency of 0.6-10% of the growth positive wells. Interestingly,very few SdrG ELISA positive hybridomas were also positive by Biacoreanalysis and whole cell bacterial binding by flow cytometry. Generally,Biacore negative, SdrG ELISA positive clones were negative in the wholecell binding flow cytometry assay. Examples of these observations areshown in Table III. TABLE III Representative Examples of HybridomaSupernatants From Fusions in Table II Immunization ELISA Data BiacoreFlow Cytometric S. epi. Fusion-Clone Antigen (SdrGN1N2N3) AnalysisStaining 41-19 SdrGN1N2N3 0.276 − − 41-75 SdrGN1N2N3 0.831 + + 41-129SdrGN1N2N3 1.195 + − 41-206 SdrGN1N2N3 0.780 + + 41-211 SdrGN1N2N30.731 + + 42-31 SdrGN1N2N3 0.537 + − 42-76 SdrGN1N2N3 0.266 − − 59-59SdrGN2N3 0.459 + + 62-27 SdrGN2N3 0.555 − ND 62-17 SdrGN2N3 0.640 + −62-02 SdrGN2N3 0.437 + − 63-06 SdrGN2N3 0.717 + + 64-03 SdrGN1N2N30.873 + + 64-04 SdrGN1N2N3 0.700 + + 64-07 SdrGN1N2N3 0.742 + + 80-01SdrGN2N3 0.671 − + 80-02 SdrGN2N3 0.602 + + 81-01 SdrGN2N3 0.664 + +81-02 SdrGN2N3 0.743 + + 81-03 SdrGN2N3 0.512 + + 82-05 SdrGTR2 0.892 +ND 83-02 SdrGTR2 0.753 + ND 83-07 SdrGTR2 0.731 + ND 83-10 SdrGTR30.654 + ND 83-13 SdrGTR2 0.671 + ND 83-17 SdrGTR2 0.678 + ND 83-20SdrGTR2 0.631 + ND 83-21 SdrGTR2 0.564 + ND

[0078] From this analysis, a very small subpopulation of growth positivehybridoma wells that were SdrG ELISA positive, SdrG Biacore positive andflow cytometry positive on Lactococcus/SdrG were single cell cloned andcharacterized as candidates for potential efficacy against S.epidermidis infection models. Table IV shows this preliminarycharacterization. TABLE IV Single Cell Cloned and Characterized SdrGMabs. Flow Biacore Analysis Cytometric Immunization ELISA Data BindingPhase Dissociation Phase SdrG Fusion/clone Antigen (SdrGN1N2N3) (RU)(RU) Staining 41-75.3 SdrGN1N2N3 0.831 218.7 173.3 + 41-206.4 SdrGN1N2N30.899 83.3 66.4 + 41-211.3 SdrGN1N2N3 0.739 80.4 64.2 + 59-59.4 SdrGN2N30.459 19.0 8.6 + 62-23.4 SdrGN1N2N3 0.517 103.0 87.2 + 62-37.10 SdrGN2N30.425 22.5 ND + 62-71.4 SdrGN2N3 0.642 60.1 ND + 63-02.6 SdrGN2N3 0.67327.3 28.2 + 63-03 SdrGN2N3 0.621 47.4 37.1 + 63-08.4 SdrGN2N3 0.639 24.624.3 + 64-03.6 SdrGN1N2N3 0.562 29.5 30.1 + 64-04.3 SdrGN1N2N3 0.81811.6 13.4 + 64-07.3 SdrGN1N2N3 0.846 20.5 20.7 + 80-01.21 SdrGN2N3 0.6713.7 1.2 + 80-02.4 SdrGN2N3 0.602 490.7 453.6 + 81-01.12 SdrGN2N3 0.664553.3 487.0 + 81-02.1 SdrGN2N3 0.743 821.2 767.8 + 81-03.5 SdrGN2N30.512 425.4 289.8 +

Example 4 Binding Kinetics of Cloned Anti-SdrG Monoclonal Antibodies

[0079] Kinetic analysis was performed to demonstrate the diversity ofthe anti-SdrG mAbs chosen and characterized. As shown below the mAbsdiffer in there on-rate and off-rate as well as the overall affinity.

[0080] Biacore Kinetics

[0081] Kinetic analysis was performed on a Biacore 3000 using the Ligandcapture method included in the software. A GAH-F(ab)₂ chip. Theanti-SdrG mAbs were then passed over a GAM-F(ab)₂ chip, allowing bindingto the Fc portion. Varying concentrations of the SdrG protein were thenpassed over the chip surface and data collected. Using the Biacoreprovided Evaluation software (Version 3.1), k_(on) and k_(off) weremeasured and K_(A) and K_(D) were calculated. TABLE V Kinetic Analysisusing the Biacore k_(a) k_(d) K_(A) Run Association Rate; DisassociationAffinity K_(D) Disassociation Mab # Lot # msec⁻¹ Rate; sec⁻¹ Constant;M⁻1 Constant; M 59-59 R658 IAA2E2122 3.42 × 10⁴ 1.38 × 10⁻² 2.48 × 10⁶4.04 × 10⁻⁷ 41-075 R224 Sup 3.78 × 10⁵ 2.72 × 10⁻³ 1.39 × 10⁸ 7.16 ×10⁻⁹ 41-206 R228 Sup 9.87 × 10⁴ 2.53 × 10⁻³ 3.97 × 10⁷ 2.56 × 10⁻⁸ 62-71R663 IAA2C2049 6.07 × 10⁵ 2.41 × 10⁻² 2.52 × 10⁷ 3.97 × 10⁻⁸ 63-02 R661IAA2B2030 3.28 × 10⁴ 5.03 × 10⁻⁴ 6.52 × 10⁷ 1.53 × 10⁻⁸ 64-03 R660IAA2C2058 5.43 × 10⁴ 2.84 × 10⁻⁴ 1.91 × 10⁸ 5.23 × 10⁻⁹ 64-04 R669IAA2J2260 9.94 × 10⁴ 1.20 × 10⁻⁴ 8.28 × 10⁸ 1.21 × 10⁻⁹ 64-07 R670IAA2D2080 2.57 × 10⁴ 5.58 × 10⁻⁴ 4.60 × 10⁷ 2.17 × 10⁻⁸

Example 5 Binding of Cloned Anti-SdrG Monoclonal Antibodies to Whole S.epidermidis Bacteria

[0082] To determine that the anti-SdrG mAbs generated and selected withrecombinant SdrG cross-reacted with native SdrG expressed onCoagulase-negative Staph. bacteria flow cytometric analysis was used. Inall cases the mAbs recognized the SdrG expressed on L. lactis, butvaried in reactivity to HB and F40802.

[0083] Binding to Whole Bacteria

[0084] Bacterial samples (HB, F40802 or SdrG/lactococcus) werecollected, washed and incubated with Mab or PBS alone (control) at aconcentration of 2 mg/ml after blocking with rabbit IgG (50 mg/ml).Following incubation with antibody, bacterial cells were incubated withGoat-F_((ab′)2)-Anti-Mouse-F_((ab′)2)-FITC which served as the detectionantibody. After antibody labeling, bacterial cells were aspiratedthrough the FACScaliber flow cytometer to analyze fluorescence emission(excitation: 488, emission: 570). For each bacterial strain, 10,000events were collected and measured. Units were determined by multiplyingthe percent of the gated positive events by the geometric mean of thestained population. TABLE VI Flow Cytometric Straining of WholeCoagulase-Negative Stphylococcus Bacteria Purified Clone L. lactis SdrGHB F40802 41-75.3 98,777 2,693 3,741 41-206.4 121,237 1,766 2,03241-211.3 90,621 1,648 2,092 59-59.4 29,976 6 1,509 64-03.6 24,108 1,032982 64-04.3 23,892 1,362 1,015 64-07.3 24,893 799 837 80-01.21 2,665 1625

Example 6 Inhibition of SdrG Binding to Fibrinogen

[0085] A number of the selected anti-SdrG mAbs of high affinity alsodisplayed the ability to inhibit human fibrinogen or the p-fibrinogenpeptide fragment binding to the SdrG MSCRAMM. This inhibition wascharacterized using a number of assays described below. This datasuggests that is may be possible to inhibit the adhesive properties ofthe SdrG MSCRAMM to human fibrinogen.

[0086] Biacore Analysis—mAb Binding to SdrG Coupled with Inhibition ofSdrG-Fibrinogen Binding

[0087] Throughout the analysis, the flow rate remained constant at 10ml/min. Prior to the SdrGN1N2N3 or SdrGN2N3 injection, test antibody wasadsorbed to the chip via RAM-Fc binding. At time 0, SdrG (N1N2 orN1N2N3) at a concentration of 30 mg/ml was injected over the chip for 3min followed by 2 minutes of dissociation. This phase of the analysismeasured the relative association and disassociation kinetics of theMab/SdrG interaction. In the second phase of the analysis, the abilityof the Mab bound SdrG to interact and bind fibrinogen was measured.Fibrinogen at a concentration of 100 mg/ml was injected over the chipand after 3 minutes a report point is taken. Examples of binding of someof the mAbs in accordance with the invention is shown in FIG. 1.

[0088] Biacore Analysis—mAb Inhibition of SdrG binding to theβ-Fibrinogen Peptide Coupled to the Chip

[0089] The precise binding site for SdrG on the fibrinogen molecule hasbeen localized to the N-terminal portion of the β-chain. For furtheranalysis and characterization, we synthesized a peptide containing thissite with the addition of an N-terminal Cysteine residue, the sequencebeing:

[0090] CNEEGFFSARGHRPLD (SEQ ID NO:7)

[0091] The β-Fibrinogen peptide is thiol-coupled to a research grade CM5chip (Biacore) through the N-terminal cysteine according to theprocedures detailed by Biacore. SdrG protein (30 μg/ml; full A-domain)is mixed with varying concentrations of mAb (90 μg/ml to 0.7 μg/ml) at a1:1 ratio. The mixture was incubated at room temperature for 20 minutesand then passed over the β-Fibrinogen peptide chip and level of bindingwas measured. SdrG diluted 1:1 with buffer served as maximal SdrGbinding, and incubation a non-SdrG mAb served as a negative control.Non-inhibitors should cause a large increase (above maximal SdrGbinding) in Resonance Units (RUs) due to the large density of theSdrG/mAb complex binding to the peptide. Alternatively, inhibitorsshould reduce the level of binding below the maximal SdrG. Percentbinding was determined as follows: Raw data, in terms of RUs, aredivided by the SdrG control level multiplied by 100. Therefore SdrG withno mAb was always be 100% for a given experiment, allowing forcomparisons between runs. Examples of mAbs in accordance with theinvention showing the inhibition of SdrG Binding to the β-Fibrinogenpeptide on the Biacore Chip is shown in FIG. 2.

[0092] ELISA-Based Protein Inhibition

[0093] Immulon 2-HB high-binding 96-well plates were coated with 1 μg/mlSdrG (amino acids 50-597) or coagulase-negative staphylococcal protein(described in Example 7) in PBS and incubated 2 hours at roomtemperature. Plates were washed and blocked with 1% BSA solution for 1hour, then washed and incubated with monoclonal antibody (eitherhybridoma supernatant or purified antibody) for 1 hour at roomtemperature. Following incubation with antibody, plates were eitherwashed or left untreated, and 20 μg/ml human fibrinogen (Enzyme ResearchLab, South Bend, Ind., USA) was added. Plates were incubated 1 hour at37° C., washed, and goat anti-fibrinogen-HRP conjugate was added.Following incubation with conjugate, plates were washed and ABTSsubstrate was added. Plates then incubated 10 minutes at roomtemperature, the reaction was stopped with addition of 10% SDS, andabsorbance was read at 405 nm. All data was analyzed using SOFTmax Prov.3.1.2. software (Molecular Devices Corp., Sunnyvale, Calif., USA).MAbs in accordance with the invention exhibiting inhibition of HumanFibrinogen Binding to SdrG by ELISA are shown in FIG. 3.

Example 7 Cross-Reactivity of Anti-SdrG Monoclonal Antibodies to OtherBacterial Proteins

[0094] To assess potential cross-reactivity with other proteins found oncoagulase-negative staphylococci, the protein described below,identified in gene bank as accession #Y17116, was cloned, expressed andpurified using methods similar to the methods described in Example 1.Interestingly, considerable cross-reactivity with this protein wasidentified with a number of the anti-SdrG mAbs of the present inventionwhich thus recognized this protein. One anti-SdrG mAb (59-59) withinhibitory activity against SdrG—fibrinogen binding however, did notcross-react and did not inhibit the binding of the protein describedbelow with fibrinogen.

[0095] The full sequence of this protein (Gen Bank #Y17116), identifiedherein as SEQ ID NO:8 is as follows:MINKKNNLLIKKKPIANKSNKYAIRKFTVGTASIVIGATLLFGLGHNEAKAEENSVQDVKDSNTDDELSDSNDQSSDEEKNDVINNNQSINTDDNNQIIKKEETNNYDGIEKRSEDRTESTTNVDENEATFLQKTPQDNTHLTEEEVKESSSVESSNSSIDTAQQPSHTTINREESVQTSDNVEDSHVSDFANSKIKESNTESGKEENTIEQPNKVKEDSTTSQPSGYTNIDEKISNQDELLNLPINEYENKARPLSTTSAQPSIKRVTVNQLAAEQGSNVNHLIKVTDQSITEGYDDSEGVIKAHDAENLIYDVTFEVDDKVKSGDTMTVDIDKNTVPSDLTDSFTIPKIKDNSGEIIATGTYDNKNKQITYTFTDYVDKYENIKAHLKLTSYIDKSKVPNNNTKLDVEYKTALSSVNKTITVEYQRPNENRTANLQSMFTNIDTKNHTVEQTIYINPLRYSAKETNVNISGNGDEGSTIIDDSTIIKVYKVGDNQNLPDSNRIYDYSEYEDVTNDDYAQLGNNNDVNINFGNIDSPYIIKVISKYDPNKDDYTTIQQTVTMQTTINEYTGEFRTASYDNTIAFSTSSGQGQGDLPPEKTYKIGDYVWEDVDKDGIQNTNDNEKPLSNVLVTLTYPDGTSKSVRTDEDGKYQFDGLKNGLTYKITFETPEGYTPTLKHSGTNPALDSEGNSVWVTINGQDDMTIDSGFYQTPKYSLGNYVWYDTNKDGIQGDDEKGISGVKVTLKDENGNIISTTTTDENGKYQFDNLNSGNYIVHFDKPSGMTQITTDSGDDDEQDADGEEVHVTITDHDDFSIDNGYYDDESDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSVSDSDSDSDSDSGSDSDSDSDSDSDNDSDLGNSSDKSTKDKLPDTGANEDYGSKGTLLGTLFAGLGALLLGKRRKNRKNKN

[0096] The following amino acid sequence was also tested:

[0097] Amino Acid Sequence (60-608) (SEQ ID NO:9)EENSVQDVKDSNTDDELSDSNDQSSDEEENDVINNNQSINSDDNNQINKKEETNNNDGIEKSSEDRTESTTNVDENEATFLQKSPQDNTHLTEEEVKEPSSVESSNSSIDTAQQPSHTTINREESVQTSDNVEDSHVSDFANSKIKESNTESGKEENTIEQPNKVKEDSTTSQPSGYTNIDEKISNQDELLNLPINEYENKARPLSTTSAQPSIKRVTVNQLAAEQGSNVNHLIKVTDQSITEGYDDSEGVIKAHDAENLIYDVIFEVDDKVKSGDTMTVDIDKNTVPSDLTDSFTIPKIKDNSGEIIATGTYDNKNKQITYTFTDYVDKYENIKAHLKLTSYIDKSKVPNNNTKLDVEYKTALSSVNKTITVEYQRPNENRTANLQSMFTNIDTKNHTVEQTIYINPLRYSAKETNVNISGNGDEGSTIIDDSTIIKVYKVGDNQNLPDSNRIYDYSEYEDVTNDDYAQLGNNNDVNINFGNIDSPYIIKVISKYDPNKDDYTTIQQTVTMQTTINEYTGEFRTASYDNTIAFSTSSGQGQGDLPPEK

[0098] In accordance with the invention, monoclonal and polyclonalantibodies can thus be raised which recognize the sequences set forthabove.

[0099] Test results of ELISA-based mAb cross-reactivity are set forth inTable VII below: TABLE VII ELISA-Based mAb Cross-reactivity PurifiedClone SdrG N1N2N3 SdrG N2N3 Gen Bank #Y17116 41-75.3 0.90 + 0.8141-206.4 0.78 + 0.76 41-211.3 0.73 + 0.65 59-59.4 0.59 + 0.11 64-03.60.87 + 0.80 64-04.3 0.70 + 0.68 64-07.3 0.74 + 0.67 80-01.21 0.67 + 0.67

[0100] The results of the tests of mAb inhibition of human fibrinogenbinding to Gen Bank protein of Accession No. Y17116 are shown in FIG. 4.

Example 8 In Vivo Based Therapeutic Activity

[0101] A number of anti-SdrG mAbs in accordance with the invention weretested for efficacy in in vivo animal models to demonstrate theirpotential utility as therapeutics.

[0102] Rodent Model of S. epidermidis Infection

[0103] Timed pregnant (13-15 day) Sprague-Dawley rats were purchasedfrom Taconic Farms, (Germantown, N.Y.). 3-6 day old newborn rats wereadministered 0.35 mg of monoclonal antibody by a single intraperitoneal(IP) injection. Twenty hours following antibody administration, thenewborn rats were challenged with an (IP) injection of 2×10⁸ CFU S.epidermidis strain HB. The survival of the animals was then followed forseven days. Kaplan-Meier analysis of survival curves was performed andsignificance was tested using a log rank test (Mantel-Haenszel Test).The test results are shown below:

[0104] Sex, Species, Number, Age, Weight and Source: Species Strain SexNumber Age Weight Source Rat Sprague Male/ 112 4-5 days 9-16 CharlesDawley Female grams River

[0105] Test Groups: TREATMENT CHALLENGE Group No. of Dose Volume/Route/Time Volume/ # Pups Antibody (mg) Frequency Point Bacteria Dose Route 110 41-211 0.35 mg 0.20 ml/i.p./once S. Epidermidis 0.20 ml/i.p. 2 1041-075 0.35 mg 0.20 ml/i.p./once 3 10 41-206 0.35 mg 0.20 ml/i.p./once 410 CRL-1771 0.35 mg 0.20 ml/i.p./once

[0106] The results of the suckling Rat Pup Challenge Model of aCoagulase-Negative Staphylococcal (S. epidermidis) Infection are shownin FIG. 5.

[0107] Description of Antibody Test Reagents:

[0108] SdrG 41-211.3 Monoclonal Antibody, INH-M01023 (LN: IAA211454)

[0109] The 41-211.3 monoclonal antibody (IgG₁ subtype) was purified fromserum free hybridoma culture medium using protein G affinitychromatography. The material was reported to be at a concentration of10.4 mg/ml with an endotoxin concentration of <0.12 EU/mg of protein.The material was stored refrigerated at 4° C. On the day of injection,the material will be diluted to 1.75 mg/ml and 0.2 ml will beadministered via an intraperitoneal injection to the appropriate groupof animals. The final dose that will be administered will be 0.35 mg ofIgG.

[0110] SdrG 41-075.3 Monoclonal Antibody, INH-M01024 (LN: IAA211447)

[0111] The 41-075.3 monoclonal antibody (IgG₁ subtype) was purified fromserum free hybridoma culture medium using protein G affinitychromatography. The material was reported to be at a concentration of7.6 mg/ml with an endotoxin concentration of <0.12 EU/mg of protein. Thematerial was stored refrigerated at 4° C. On the day of injection, thematerial will be diluted to 1.75 mg/ml and 0.2 ml will be administeredvia an intraperitoneal injection to the appropriate group of animals.The final dose administered was 0.35 mg of IgG.

[0112] SdrG 41-206.4 Monoclonal Antibody, INH-M01025 (LN: IAA211448)

[0113] The 41-206.4 monoclonal antibody (IgG₁ subtype) was purified fromserum free hybridoma culture medium using protein G affinitychromatography. The material was reported to be at a concentration of8.9 mg/ml with an endotoxin concentration <0.12 EU/mg of protein. Thematerial was stored refrigerated at 4° C. On the day of injection, thematerial will be diluted to 1.75 mg/ml and 0.2 ml will be administeredvia an intraperitoneal injection to the appropriate group of animals.The final dose administered was 0.35 mg of IgG.

[0114] Control CRL1771 Monoclonal Antibody, INH-M000029 (LN: IM2G1381)

[0115] The CRL 1771 monoclonal antibody (IgG₁ subtype) was purified fromserum free hybridoma culture medium using protein G affinitychromatography. The material was reported to be at a concentration of6.6 mg/ml with an endotoxin concentration of <3.0 EU/mg of protein. Thematerial was stored refrigerated at 4° C. On the day of injection, thematerial will be diluted 1.75 mg/ml and 0.2 ml will be administered viaan intraperitoneal injection to the appropriate group of animals. Thefinal dose administered was 0.35 mg of IgG.

[0116] Rat Model of Central Venous Catheter (CVC) Associated Infection

[0117] 8-9 week old male Sprague-Dawley rats were purchased from CharlesRiver Laboratories (Raleigh, N.C.). A sterile polyethylene/siliconcatheter (catheter body-polyethylene: 0.011″ id, 0.024″ od; cathetertip-silicon rubber: 0.012″ id, 0.025″ od) was surgically placed in thejugular vein and the catheter tip was advanced into the superior venacava. The catheter remained in place and was kept patent throughout thestudy. Monoclonal antibodies were administered IV through the catheterat a dose of 20 mg/kg. 24 hours later, 5×10³ CFU of methicillinresistant S. epidermidis MRSE (Strain 899) were introduced via thecatheter. Day 7 post-challenge, the animals were sacrificed and caudalvena cava blood, kidneys and catheter associated tissues were harvested.The MRSE colony forming units present in the tissue samples weremeasured by quantitative plating. Statistical analysis of the incidenceof infection across groups was performed using Fisher's Exact Test.Statistical Analysis of quantitative differences in CFU between groupswas performed using the Kruskal-Wallis Test with Dunn's multiplecomparison post-test.

[0118] Description of Antibody Test Reagents:

[0119] SdrG 59-59.4 Monoclonal Antibody, INH-M02001 (LN: IAA2B2032)

[0120] The 41-211.3 monoclonal antibody (IgG₁ subtype) was purified fromserum free hybridoma culture medium using protein G affinitychromatography. The material was reported to be at a concentration of8.2 mg/ml with an endotoxin concentration of <0.12 EU/mg of protein. Thematerial was stored refrigerated at 4° C. On the day of injection, thematerial was administered via the catheter for a final dose 20 mg/kg ofIgG.

[0121] SdrG 64-03.6 Monoclonal Antibody, INH-M02008 (LN: IAA2C2058)

[0122] The 41-075.3 monoclonal antibody (IgG₁ subtype) was purified fromserum free hybridoma culture medium using protein G affinitychromatography. The material was reported to be at a concentration of 11mg/ml with an endotoxin concentration of <0.12 EU/mg of protein. Thematerial was stored refrigerated at 4° C. On the day of injection, thematerial was administered via the catheter for a final dose 20 mg/kg ofIgG.

[0123] Control CRL1771 Monoclonal Antibody, INH-M000029 (LN: IAA2G1381)

[0124] The CRL 1771 monoclonal antibody (IgG₁ subtype) was purified fromserum free hybridoma culture medium using protein G affinitychromatography. The material was reported to be at a concentration of6.6 mg/ml with an endotoxin concentration of <3.0 EU/mg of protein. Thematerial was stored refrigerated at 4° C. On the day of injection, thematerial was administered via the catheter for a final dose 20 mg/kg ofIgG.

[0125] Test results showing the central venous catheter (CVC) associatedinfection model of a coagulase-negative Staphylococcal (S. epidermidis)Infection at Day 7 are shown in FIG. 6.

1 9 1 1680 DNA Staphylococcus epidermidis 1 atgagaggat cgcatcaccatcaccatcac ggatccgagg agaatacagt acaagacgtt 60 aaagattcga atatggatgatgaattatca gatagcaatg atcagtccag taatgaagaa 120 aagaatgatg taatcaataatagtcagtca ataaacaccg atgatgataa ccaaataaaa 180 aaagaagaaa cgaatagcaacgatgccata gaaaatcgct ctaaagatat aacacagtca 240 acaacaaatg tagatgaaaacgaagcaaca tttttacaaa agacccctca agataatact 300 cagcttaaag aagaagtggtaaaagaaccc tcatcagtcg aatcctcaaa ttcatcaatg 360 gatactgccc aacaaccatctcatacaaca ataaatagtg aagcatctat tcaaacaagt 420 gataatgaag aaaattcccgcgtatcagat tttgctaact ctaaaataat agagagtaac 480 actgaatcca ataaagaagagaatactata gagcaaccta acaaagtaag agaagattca 540 ataacaagtc aaccgtctagctataaaaat atagatgaaa aaatttcaaa tcaagatgag 600 ttattaaatt taccaataaatgaatatgaa aataaggtta gaccgttatc tacaacatct 660 gcccaaccat cgagtaagcgtgtaaccgta aatcaattag cggcagaaca aggttcgaat 720 gttaatcatt taattaaagttactgatcaa agtattactg aaggatatga tgatagtgat 780 ggtattatta aagcacatgatgctgaaaac ttaatctatg atgtaacttt tgaagtagat 840 gataaggtga aatctggtgatacgatgaca gtgaatatag ataagaatac agttccatca 900 gatttaaccg atagttttgcaataccaaaa ataaaagata attctggaga aatcatcgct 960 acaggtactt atgacaacacaaataaacaa attacctaca cttttacaga ttatgtagat 1020 aaatatgaaa atattaaagcgcaccttaaa ttaacatcat acattgataa atcaaaggtt 1080 ccaaataata acactaagttagatgtagaa tataagacgg ccctttcatc agtaaataaa 1140 acaattacgg ttgaatatcaaaaacctaac gaaaatcgga ctgctaacct tcaaagtatg 1200 ttcacaaaca tagatacgaaaaaccataca gttgagcaaa cgatttatat taaccctctt 1260 cgttattcag ccaaagaaacaaatgtaaat atttcaggga atggcgatga aggttcaaca 1320 attatcgacg atagtacaatcattaaagtt tataaggttg gagataatca aaatttacca 1380 gatagtaaca gaatttatgattacagtgaa tatgaagatg tcacaaatga tgattatgcc 1440 caattaggaa ataataatgacgtgaatatt aattttggta atatagattc accatatatt 1500 attaaagtta ttagtaaatatgaccctaat aaggacgatt acacgacgat acagcaaact 1560 gtgacaatgc aaacgactataaatgagtat actggtgagt ttagaacagc atcctatgat 1620 aatacaattg ctttctctacaagttcaggt caaggacaag gtgacttgcc tcctgaaaaa 1680 2 560 PRTStaphylococcus epidermidis 2 Met Arg Gly Ser His His His His His His GlySer Glu Glu Asn Thr 1 5 10 15 Val Gln Asp Val Lys Asp Ser Asn Met AspAsp Glu Leu Ser Asp Ser 20 25 30 Asn Asp Gln Ser Ser Asn Glu Glu Lys AsnAsp Val Ile Asn Asn Ser 35 40 45 Gln Ser Ile Asn Thr Asp Asp Asp Asn GlnIle Lys Lys Glu Glu Thr 50 55 60 Asn Ser Asn Asp Ala Ile Glu Asn Arg SerLys Asp Ile Thr Gln Ser 65 70 75 80 Thr Thr Asn Val Asp Glu Asn Glu AlaThr Phe Leu Gln Lys Thr Pro 85 90 95 Gln Asp Asn Thr Gln Leu Lys Glu GluVal Val Lys Glu Pro Ser Ser 100 105 110 Val Glu Ser Ser Asn Ser Ser MetAsp Thr Ala Gln Gln Pro Ser His 115 120 125 Thr Thr Ile Asn Ser Glu AlaSer Ile Gln Thr Ser Asp Asn Glu Glu 130 135 140 Asn Ser Arg Val Ser AspPhe Ala Asn Ser Lys Ile Ile Glu Ser Asn 145 150 155 160 Thr Glu Ser AsnLys Glu Glu Asn Thr Ile Glu Gln Pro Asn Lys Val 165 170 175 Arg Glu AspSer Ile Thr Ser Gln Pro Ser Ser Tyr Lys Asn Ile Asp 180 185 190 Glu LysIle Ser Asn Gln Asp Glu Leu Leu Asn Leu Pro Ile Asn Glu 195 200 205 TyrGlu Asn Lys Val Arg Pro Leu Ser Thr Thr Ser Ala Gln Pro Ser 210 215 220Ser Lys Arg Val Thr Val Asn Gln Leu Ala Ala Glu Gln Gly Ser Asn 225 230235 240 Val Asn His Leu Ile Lys Val Thr Asp Gln Ser Ile Thr Glu Gly Tyr245 250 255 Asp Asp Ser Asp Gly Ile Ile Lys Ala His Asp Ala Glu Asn LeuIle 260 265 270 Tyr Asp Val Thr Phe Glu Val Asp Asp Lys Val Lys Ser GlyAsp Thr 275 280 285 Met Thr Val Asn Ile Asp Lys Asn Thr Val Pro Ser AspLeu Thr Asp 290 295 300 Ser Phe Ala Ile Pro Lys Ile Lys Asp Asn Ser GlyGlu Ile Ile Ala 305 310 315 320 Thr Gly Thr Tyr Asp Asn Thr Asn Lys GlnIle Thr Tyr Thr Phe Thr 325 330 335 Asp Tyr Val Asp Lys Tyr Glu Asn IleLys Ala His Leu Lys Leu Thr 340 345 350 Ser Tyr Ile Asp Lys Ser Lys ValPro Asn Asn Asn Thr Lys Leu Asp 355 360 365 Val Glu Tyr Lys Thr Ala LeuSer Ser Val Asn Lys Thr Ile Thr Val 370 375 380 Glu Tyr Gln Lys Pro AsnGlu Asn Arg Thr Ala Asn Leu Gln Ser Met 385 390 395 400 Phe Thr Asn IleAsp Thr Lys Asn His Thr Val Glu Gln Thr Ile Tyr 405 410 415 Ile Asn ProLeu Arg Tyr Ser Ala Lys Glu Thr Asn Val Asn Ile Ser 420 425 430 Gly AsnGly Asp Glu Gly Ser Thr Ile Ile Asp Asp Ser Thr Ile Ile 435 440 445 LysVal Tyr Lys Val Gly Asp Asn Gln Asn Leu Pro Asp Ser Asn Arg 450 455 460Ile Tyr Asp Tyr Ser Glu Tyr Glu Asp Val Thr Asn Asp Asp Tyr Ala 465 470475 480 Gln Leu Gly Asn Asn Asn Asp Val Asn Ile Asn Phe Gly Asn Ile Asp485 490 495 Ser Pro Tyr Ile Ile Lys Val Ile Ser Lys Tyr Asp Pro Asn LysAsp 500 505 510 Asp Tyr Thr Thr Ile Gln Gln Thr Val Thr Met Gln Thr ThrIle Asn 515 520 525 Glu Tyr Thr Gly Glu Phe Arg Thr Ala Ser Tyr Asp AsnThr Ile Ala 530 535 540 Phe Ser Thr Ser Ser Gly Gln Gly Gln Gly Asp LeuPro Pro Glu Lys 545 550 555 560 3 1030 DNA Staphylococcus epidermidis 3atgagaggat cgcatcacca tcaccatcac ggatctctgg ttcctagggg atccgaacaa 60ggttcgaatg ttaatcattt aattaaagtt actgatcaaa gtattactga aggatatgat 120gatagtgatg gtattattaa agcacatgat gctgaaaact taatctatga tgtaactttt 180gaagtagatg ataaggtgaa atctggtgat acgatgacag tgaatataga taagaataca 240gttccatcag atttaaccga tagttttgca ataccaaaaa taaaagataa ttctggagaa 300atcatcgcta caggtactta tgacaacaca aataaacaaa ttacctacac ttttacagat 360tatgtagata aatatgaaaa tattaaagcg caccttaaat taacatcata cattgataaa 420tcaaaggttc caaataataa cactaagtta gatgtagaat ataagacggc cctttcatca 480gtaaataaaa caattacggt tgaatatcaa aaacctaacg aaaatcggac tgctaacctt 540caaagtatgt tcacaaacat agatacgaaa aaccatacag ttgagcaaac gatttatatt 600aaccctcttc gttattcagc caaagaaaca aatgtaaata tttcagggaa tggcgatgaa 660ggttcaacaa ttatcgacga tagtacaatc attaaagttt ataaggttgg agataatcaa 720aatttaccag atagtaacag aatttatgat tacagtgaat atgaagatgt cacaaatgat 780gattatgccc aattaggaaa taataatgac gtgaatatta attttggtaa tatagattca 840ccatatatta ttaaagttat tagtaaatat gaccctaata aggacgatta cacgacgata 900cagcaaactg tgacaatgca aacgactata aatgagtata ctggtgagtt tagaacagca 960tcctatgata atacaattgc tttctctaca agttcaggtc aaggacaagg tgacttgcct 1020cctgaaaaat 1030 4 343 PRT Staphylococcus epidermidis 4 Met Arg Gly SerHis His His His His His Gly Ser Leu Val Pro Arg 1 5 10 15 Gly Ser GluGln Gly Ser Asn Val Asn His Leu Ile Lys Val Thr Asp 20 25 30 Gln Ser IleThr Glu Gly Tyr Asp Asp Ser Asp Gly Ile Ile Lys Ala 35 40 45 His Asp AlaGlu Asn Leu Ile Tyr Asp Val Thr Phe Glu Val Asp Asp 50 55 60 Lys Val LysSer Gly Asp Thr Met Thr Val Asn Ile Asp Lys Asn Thr 65 70 75 80 Val ProSer Asp Leu Thr Asp Ser Phe Ala Ile Pro Lys Ile Lys Asp 85 90 95 Asn SerGly Glu Ile Ile Ala Thr Gly Thr Tyr Asp Asn Thr Asn Lys 100 105 110 GlnIle Thr Tyr Thr Phe Thr Asp Tyr Val Asp Lys Tyr Glu Asn Ile 115 120 125Lys Ala His Leu Lys Leu Thr Ser Tyr Ile Asp Lys Ser Lys Val Pro 130 135140 Asn Asn Asn Thr Lys Leu Asp Val Glu Tyr Lys Thr Ala Leu Ser Ser 145150 155 160 Val Asn Lys Thr Ile Thr Val Glu Tyr Gln Lys Pro Asn Glu AsnArg 165 170 175 Thr Ala Asn Leu Gln Ser Met Phe Thr Asn Ile Asp Thr LysAsn His 180 185 190 Thr Val Glu Gln Thr Ile Tyr Ile Asn Pro Leu Arg TyrSer Ala Lys 195 200 205 Glu Thr Asn Val Asn Ile Ser Gly Asn Gly Asp GluGly Ser Thr Ile 210 215 220 Ile Asp Asp Ser Thr Ile Ile Lys Val Tyr LysVal Gly Asp Asn Gln 225 230 235 240 Asn Leu Pro Asp Ser Asn Arg Ile TyrAsp Tyr Ser Glu Tyr Glu Asp 245 250 255 Val Thr Asn Asp Asp Tyr Ala GlnLeu Gly Asn Asn Asn Asp Val Asn 260 265 270 Ile Asn Phe Gly Asn Ile AspSer Pro Tyr Ile Ile Lys Val Ile Ser 275 280 285 Lys Tyr Asp Pro Asn LysAsp Asp Tyr Thr Thr Ile Gln Gln Thr Val 290 295 300 Thr Met Gln Thr ThrIle Asn Glu Tyr Thr Gly Glu Phe Arg Thr Ala 305 310 315 320 Ser Tyr AspAsn Thr Ile Ala Phe Ser Thr Ser Ser Gly Gln Gly Gln 325 330 335 Gly AspLeu Pro Pro Glu Lys 340 5 951 DNA Staphylococcus epidermidis 5atgagaggat cgcatcacca tcaccatcac ggatccgaac aaggttcgaa tgttaatcat 60ttaattaaag ttactgatca aagtattact gaaggatatg atgatagtga tggtattatt 120aaagcacatg atgctgaaaa cttaatctat gatgtaactt ttgaagtaga tgataaggtg 180aaatctggtg atacgatgac agtgaatata gataagaata cagttccatc agatttaacc 240gatagttttg caataccaaa aataaaagat aattctggag aaatcatcgc tacaggtact 300tatgacaaca caaataaaca aattacctac acttttacag attatgtaga taaatatgaa 360aatattaaag cgcaccttaa attaacatca tacattgata aatcaaaggt tccaaataat 420aacactaagt tagatgtaga atataagacg gccctttcat cagtaaataa aacaattacg 480gttgaatatc aaaaacctaa cgaaaatcgg actgctaacc ttcaaagtat gttcacaaac 540atagatacga aaaaccatac agttgagcaa acgatttata ttaaccctct tcgttattca 600gccaaagaaa caaatgtaaa tatttcaggg aatggcgatg aaggttcaac aattatcgac 660gatagtacaa tcattaaagt ttataaggtt ggagataatc aaaatttacc agatagtaac 720agaatttatg attacagtga atatgaagat gtcacaaatg atgattatgc ccaattagga 780aataataatg acgtgaatat taattttggt aatatagatt caccatatat tattaaagtt 840attagtaaat atgaccctaa taaggacgat tacacgacga tacagcaaac tgtgacaatg 900caaacgacta taaatgagta tactggtgag tttagaacag catcctattg a 951 6 316 PRTStaphylococcus epidermidis 6 Met Arg Gly Ser His His His His His His GlySer Glu Gln Gly Ser 1 5 10 15 Asn Val Asn His Leu Ile Lys Val Thr AspGln Ser Ile Thr Glu Gly 20 25 30 Tyr Asp Asp Ser Asp Gly Ile Ile Lys AlaHis Asp Ala Glu Asn Leu 35 40 45 Ile Tyr Asp Val Thr Phe Glu Val Asp AspLys Val Lys Ser Gly Asp 50 55 60 Thr Met Thr Val Asn Ile Asp Lys Asn ThrVal Pro Ser Asp Leu Thr 65 70 75 80 Asp Ser Phe Ala Ile Pro Lys Ile LysAsp Asn Ser Gly Glu Ile Ile 85 90 95 Ala Thr Gly Thr Tyr Asp Asn Thr AsnLys Gln Ile Thr Tyr Thr Phe 100 105 110 Thr Asp Tyr Val Asp Lys Tyr GluAsn Ile Lys Ala His Leu Lys Leu 115 120 125 Thr Ser Tyr Ile Asp Lys SerLys Val Pro Asn Asn Asn Thr Lys Leu 130 135 140 Asp Val Glu Tyr Lys ThrAla Leu Ser Ser Val Asn Lys Thr Ile Thr 145 150 155 160 Val Glu Tyr GlnLys Pro Asn Glu Asn Arg Thr Ala Asn Leu Gln Ser 165 170 175 Met Phe ThrAsn Ile Asp Thr Lys Asn His Thr Val Glu Gln Thr Ile 180 185 190 Tyr IleAsn Pro Leu Arg Tyr Ser Ala Lys Glu Thr Asn Val Asn Ile 195 200 205 SerGly Asn Gly Asp Glu Gly Ser Thr Ile Ile Asp Asp Ser Thr Ile 210 215 220Ile Lys Val Tyr Lys Val Gly Asp Asn Gln Asn Leu Pro Asp Ser Asn 225 230235 240 Arg Ile Tyr Asp Tyr Ser Glu Tyr Glu Asp Val Thr Asn Asp Asp Tyr245 250 255 Ala Gln Leu Gly Asn Asn Asn Asp Val Asn Ile Asn Phe Gly AsnIle 260 265 270 Asp Ser Pro Tyr Ile Ile Lys Val Ile Ser Lys Tyr Asp ProAsn Lys 275 280 285 Asp Asp Tyr Thr Thr Ile Gln Gln Thr Val Thr Met GlnThr Thr Ile 290 295 300 Asn Glu Tyr Thr Gly Glu Phe Arg Thr Ala Ser Tyr305 310 315 7 16 PRT Staphylococcus epidermidis 7 Cys Asn Glu Glu GlyPhe Phe Ser Ala Arg Gly His Arg Pro Leu Asp 1 5 10 15 8 1092 PRTStaphylococcus epidermidis 8 Met Ile Asn Lys Lys Asn Asn Leu Leu Thr LysLys Lys Pro Ile Ala 1 5 10 15 Asn Lys Ser Asn Lys Tyr Ala Ile Arg LysPhe Thr Val Gly Thr Ala 20 25 30 Ser Ile Val Ile Gly Ala Thr Leu Leu PheGly Leu Gly His Asn Glu 35 40 45 Ala Lys Ala Glu Glu Asn Ser Val Gln AspVal Lys Asp Ser Asn Thr 50 55 60 Asp Asp Glu Leu Ser Asp Ser Asn Asp GlnSer Ser Asp Glu Glu Lys 65 70 75 80 Asn Asp Val Ile Asn Asn Asn Gln SerIle Asn Thr Asp Asp Asn Asn 85 90 95 Gln Ile Ile Lys Lys Glu Glu Thr AsnAsn Tyr Asp Gly Ile Glu Lys 100 105 110 Arg Ser Glu Asp Arg Thr Glu SerThr Thr Asn Val Asp Glu Asn Glu 115 120 125 Ala Thr Phe Leu Gln Lys ThrPro Gln Asp Asn Thr His Leu Thr Glu 130 135 140 Glu Glu Val Lys Glu SerSer Ser Val Glu Ser Ser Asn Ser Ser Ile 145 150 155 160 Asp Thr Ala GlnGln Pro Ser His Thr Thr Ile Asn Arg Glu Glu Ser 165 170 175 Val Gln ThrSer Asp Asn Val Glu Asp Ser His Val Ser Asp Phe Ala 180 185 190 Asn SerLys Ile Lys Glu Ser Asn Thr Glu Ser Gly Lys Glu Glu Asn 195 200 205 ThrIle Glu Gln Pro Asn Lys Val Lys Glu Asp Ser Thr Thr Ser Gln 210 215 220Pro Ser Gly Tyr Thr Asn Ile Asp Glu Lys Ile Ser Asn Gln Asp Glu 225 230235 240 Leu Leu Asn Leu Pro Ile Asn Glu Tyr Glu Asn Lys Ala Arg Pro Leu245 250 255 Ser Thr Thr Ser Ala Gln Pro Ser Ile Lys Arg Val Thr Val AsnGln 260 265 270 Leu Ala Ala Glu Gln Gly Ser Asn Val Asn His Leu Ile LysVal Thr 275 280 285 Asp Gln Ser Ile Thr Glu Gly Tyr Asp Asp Ser Glu GlyVal Ile Lys 290 295 300 Ala His Asp Ala Glu Asn Leu Ile Tyr Asp Val ThrPhe Glu Val Asp 305 310 315 320 Asp Lys Val Lys Ser Gly Asp Thr Met ThrVal Asp Ile Asp Lys Asn 325 330 335 Thr Val Pro Ser Asp Leu Thr Asp SerPhe Thr Ile Pro Lys Ile Lys 340 345 350 Asp Asn Ser Gly Glu Ile Ile AlaThr Gly Thr Tyr Asp Asn Lys Asn 355 360 365 Lys Gln Ile Thr Tyr Thr PheThr Asp Tyr Val Asp Lys Tyr Glu Asn 370 375 380 Ile Lys Ala His Leu LysLeu Thr Ser Tyr Ile Asp Lys Ser Lys Val 385 390 395 400 Pro Asn Asn AsnThr Lys Leu Asp Val Glu Tyr Lys Thr Ala Leu Ser 405 410 415 Ser Val AsnLys Thr Ile Thr Val Glu Tyr Gln Arg Pro Asn Glu Asn 420 425 430 Arg ThrAla Asn Leu Gln Ser Met Phe Thr Asn Ile Asp Thr Lys Asn 435 440 445 HisThr Val Glu Gln Thr Ile Tyr Ile Asn Pro Leu Arg Tyr Ser Ala 450 455 460Lys Glu Thr Asn Val Asn Ile Ser Gly Asn Gly Asp Glu Gly Ser Thr 465 470475 480 Ile Ile Asp Asp Ser Thr Ile Ile Lys Val Tyr Lys Val Gly Asp Asn485 490 495 Gln Asn Leu Pro Asp Ser Asn Arg Ile Tyr Asp Tyr Ser Glu TyrGlu 500 505 510 Asp Val Thr Asn Asp Asp Tyr Ala Gln Leu Gly Asn Asn AsnAsp Val 515 520 525 Asn Ile Asn Phe Gly Asn Ile Asp Ser Pro Tyr Ile IleLys Val Ile 530 535 540 Ser Lys Tyr Asp Pro Asn Lys Asp Asp Tyr Thr ThrIle Gln Gln Thr 545 550 555 560 Val Thr Met Gln Thr Thr Ile Asn Glu TyrThr Gly Glu Phe Arg Thr 565 570 575 Ala Ser Tyr Asp Asn Thr Ile Ala PheSer Thr Ser Ser Gly Gln Gly 580 585 590 Gln Gly Asp Leu Pro Pro Glu LysThr Tyr Lys Ile Gly Asp Tyr Val 595 600 605 Trp Glu Asp Val Asp Lys AspGly Ile Gln Asn Thr Asn Asp Asn Glu 610 615 620 Lys Pro Leu Ser Asn ValLeu Val Thr Leu Thr Tyr Pro Asp Gly Thr 625 630 635 640 Ser Lys Ser ValArg Thr Asp Glu Asp Gly Lys Tyr Gln Phe Asp Gly 645 650 655 Leu Lys AsnGly Leu Thr Tyr Lys Ile Thr Phe Glu Thr Pro Glu Gly 660 665 670 Tyr ThrPro Thr Leu Lys His Ser Gly Thr Asn Pro Ala Leu Asp Ser 675 680 685 GluGly Asn Ser Val Trp Val Thr Ile Asn Gly Gln Asp Asp Met Thr 690 695 700Ile Asp Ser Gly Phe Tyr Gln Thr Pro Lys Tyr Ser Leu Gly Asn Tyr 705 710715 720 Val Trp Tyr Asp Thr Asn Lys Asp Gly Ile Gln Gly Asp Asp Glu Lys725 730 735 Gly Ile Ser Gly Val Lys Val Thr Leu Lys Asp Glu Asn Gly AsnIle 740 745 750 Ile Ser Thr Thr Thr Thr Asp Glu Asn Gly Lys Tyr Gln PheAsp Asn 755 760 765 Leu Asn Ser Gly Asn Tyr Ile Val His Phe Asp Lys ProSer Gly Met 770 775 780 Thr Gln Thr Thr Thr Asp Ser Gly Asp Asp Asp GluGln Asp Ala Asp 785 790 795 800 Gly Glu Glu Val His Val Thr Ile Thr AspHis Asp Asp Phe Ser Ile 805 810 815 Asp Asn Gly Tyr Tyr Asp Asp Glu SerAsp Ser Asp Ser Asp Ser Asp 820 825 830 Ser Asp Ser Asp Ser Asp Ser AspSer Asp Ser Asp Ser Asp Ser Asp 835 840 845 Ser Asp Ser Asp Ser Asp SerAsp Ser Asp Ser Asp Ser Asp Ser Asp 850 855 860 Ser Asp Ser Asp Ser AspSer Asp Ser Asp Ser Asp Ser Asp Ser Asp 865 870 875 880 Ser Asp Ser AspSer Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp 885 890 895 Ser Asp SerAsp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp 900 905 910 Ser AspSer Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp 915 920 925 SerAsp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp 930 935 940Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp 945 950955 960 Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp965 970 975 Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp SerAsp 980 985 990 Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser AspSer Asp 995 1000 1005 Ser Asp Ser Val Ser Asp Ser Asp Ser Asp Ser AspSer Asp Ser 1010 1015 1020 Gly Ser Asp Ser Asp Ser Asp Ser Asp Ser AspSer Asp Asn Asp 1025 1030 1035 Ser Asp Leu Gly Asn Ser Ser Asp Lys SerThr Lys Asp Lys Leu 1040 1045 1050 Pro Asp Thr Gly Ala Asn Glu Asp TyrGly Ser Lys Gly Thr Leu 1055 1060 1065 Leu Gly Thr Leu Phe Ala Gly LeuGly Ala Leu Leu Leu Gly Lys 1070 1075 1080 Arg Arg Lys Asn Arg Lys AsnLys Asn 1085 1090 9 549 PRT Staphylococcus epidermidis 9 Glu Glu Asn SerVal Gln Asp Val Lys Asp Ser Asn Thr Asp Asp Glu 1 5 10 15 Leu Ser AspSer Asn Asp Gln Ser Ser Asp Glu Glu Glu Asn Asp Val 20 25 30 Ile Asn AsnAsn Gln Ser Ile Asn Ser Asp Asp Asn Asn Gln Ile Asn 35 40 45 Lys Lys GluGlu Thr Asn Asn Asn Asp Gly Ile Glu Lys Ser Ser Glu 50 55 60 Asp Arg ThrGlu Ser Thr Thr Asn Val Asp Glu Asn Glu Ala Thr Phe 65 70 75 80 Leu GlnLys Ser Pro Gln Asp Asn Thr His Leu Thr Glu Glu Glu Val 85 90 95 Lys GluPro Ser Ser Val Glu Ser Ser Asn Ser Ser Ile Asp Thr Ala 100 105 110 GlnGln Pro Ser His Thr Thr Ile Asn Arg Glu Glu Ser Val Gln Thr 115 120 125Ser Asp Asn Val Glu Asp Ser His Val Ser Asp Phe Ala Asn Ser Lys 130 135140 Ile Lys Glu Ser Asn Thr Glu Ser Gly Lys Glu Glu Asn Thr Ile Glu 145150 155 160 Gln Pro Asn Lys Val Lys Glu Asp Ser Thr Thr Ser Gln Pro SerGly 165 170 175 Tyr Thr Asn Ile Asp Glu Lys Ile Ser Asn Gln Asp Glu LeuLeu Asn 180 185 190 Leu Pro Ile Asn Glu Tyr Glu Asn Lys Ala Arg Pro LeuSer Thr Thr 195 200 205 Ser Ala Gln Pro Ser Ile Lys Arg Val Thr Val AsnGln Leu Ala Ala 210 215 220 Glu Gln Gly Ser Asn Val Asn His Leu Ile LysVal Thr Asp Gln Ser 225 230 235 240 Ile Thr Glu Gly Tyr Asp Asp Ser GluGly Val Ile Lys Ala His Asp 245 250 255 Ala Glu Asn Leu Ile Tyr Asp ValThr Phe Glu Val Asp Asp Lys Val 260 265 270 Lys Ser Gly Asp Thr Met ThrVal Asp Ile Asp Lys Asn Thr Val Pro 275 280 285 Ser Asp Leu Thr Asp SerPhe Thr Ile Pro Lys Ile Lys Asp Asn Ser 290 295 300 Gly Glu Ile Ile AlaThr Gly Thr Tyr Asp Asn Lys Asn Lys Gln Ile 305 310 315 320 Thr Tyr ThrPhe Thr Asp Tyr Val Asp Lys Tyr Glu Asn Ile Lys Ala 325 330 335 His LeuLys Leu Thr Ser Tyr Ile Asp Lys Ser Lys Val Pro Asn Asn 340 345 350 AsnThr Lys Leu Asp Val Glu Tyr Lys Thr Ala Leu Ser Ser Val Asn 355 360 365Lys Thr Ile Thr Val Glu Tyr Gln Arg Pro Asn Glu Asn Arg Thr Ala 370 375380 Asn Leu Gln Ser Met Phe Thr Asn Ile Asp Thr Lys Asn His Thr Val 385390 395 400 Glu Gln Thr Ile Tyr Ile Asn Pro Leu Arg Tyr Ser Ala Lys GluThr 405 410 415 Asn Val Asn Ile Ser Gly Asn Gly Asp Glu Gly Ser Thr IleIle Asp 420 425 430 Asp Ser Thr Ile Ile Lys Val Tyr Lys Val Gly Asp AsnGln Asn Leu 435 440 445 Pro Asp Ser Asn Arg Ile Tyr Asp Tyr Ser Glu TyrGlu Asp Val Thr 450 455 460 Asn Asp Asp Tyr Ala Gln Leu Gly Asn Asn AsnAsp Val Asn Ile Asn 465 470 475 480 Phe Gly Asn Ile Asp Ser Pro Tyr IleIle Lys Val Ile Ser Lys Tyr 485 490 495 Asp Pro Asn Lys Asp Asp Tyr ThrThr Ile Gln Gln Thr Val Thr Met 500 505 510 Gln Thr Thr Ile Asn Glu TyrThr Gly Glu Phe Arg Thr Ala Ser Tyr 515 520 525 Asp Asn Thr Ile Ala PheSer Thr Ser Ser Gly Gln Gly Gln Gly Asp 530 535 540 Leu Pro Pro Glu Lys545

What is claimed is:
 1. An isolated antibody that recognizes a proteinfrom S. epidermidis selected from the group consisting of SdrG N1N2N3,SdrG N2N3 and SdrG TR2.
 2. The antibody according to claim 1 wherein theantibody is a monoclonal antibody.
 3. The monoclonal antibody accordingto claim 2 wherein the antibody is of a type selected from the groupconsisting of chimeric, murine, humanized and human monoclonalantibodies.
 4. The monoclonal antibody according to claim 2 wherein theantibody is a single chain monoclonal antibody.
 5. The antibodyaccording to claim 1, wherein said antibody prevents acoagulase-negative staphylococcal infection in a human or animal.
 6. Theantibody according to claim 1, wherein said antibody inhibits binding ofstaphylococcal bacteria to fibrinogen.
 7. The antibody according toclaim 1, wherein said antibody is suitable for parenteral, oral,intranasal, subcutaneous, aerosolized or intravenous administration in ahuman or animal.
 8. The antibody according to claim 1 wherein theantibody binds to the S. epidermidis SdrG protein.
 9. The antibodyaccording to claim 1 wherein the antibody recognizes an amino acidsequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4and SEQ ID NO:6.
 10. The antibody according to claim 1 wherein theantibody recognizes an amino acid sequence encoded by a nucleic acidsequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3and SEQ ID NO:5 and degenerates thereof.
 11. Isolated antiseracontaining an antibody according to claim
 1. 12. A diagnostic kitcomprising an antibody according to claim 1 and means for detectingbinding by that antibody.
 13. A diagnostic kit according to claim 12wherein said means for detecting binding comprises a detectable labelthat is linked to said antibody.
 14. A method of treating or preventinga coagulase-negative staphylococcal infection comprising administeringto a human or animal patient an effective amount of an antibodyaccording to claim
 1. 15. A pharmaceutical composition for treating orpreventing a coagulase-negative staphylococcal comprising an effectiveamount of the antibody of claim 1 and a pharmaceutically acceptablevehicle, carrier or excipient.
 16. An isolated antibody that recognizesthe protein sequence of SEQ ID NO:8.
 17. The antibody according to claim16 wherein the antibody is a monoclonal antibody.
 18. A method oftreating or preventing a coagulase-negative staphylococcal infectioncomprising administering to a human or animal patient an effectiveamount of an antibody according to claim
 16. 19. A pharmaceuticalcomposition for treating or preventing a coagulase-negativestaphylococcal comprising an effective amount of the antibody of claim16 and a pharmaceutically acceptable vehicle, carrier or excipient. 20.An isolated antibody that recognizes the amino acid sequence of SEQ IDNO:9.
 21. The antibody according to claim 20 wherein the antibody is amonoclonal antibody.
 22. A method of treating or preventing acoagulase-negative staphylococcal infection comprising administering toa human or animal patient an effective amount of an antibody accordingto claim
 20. 23. A pharmaceutical composition for treating or preventinga coagulase-negative staphylococcal comprising an effective amount ofthe antibody of claim 20 and a pharmaceutically acceptable vehicle,carrier or excipient.
 24. An isolated S. epidermidis protein selectedfrom the group consisting of SdrG N1N2N3, SdrG N2N3 and SdrG TR2.
 25. Amethod of eliciting an immunogenic reaction in a human or animalcomprising administering to said human or animal an immunologicallyeffective amount of an isolated protein according to claim
 24. 26. Avaccine comprising an immunogenic amount of the isolated proteinaccording to claim 24 and a pharmaceutically acceptable vehicle, carrieror excipient.
 27. The isolated protein according to claim 24 wherein theprotein has an amino acid sequence selected from the group consisting ofSEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:6.
 28. The isolated proteinaccording to claim 24 wherein the protein is encoded by a nucleic acidsequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3and SEQ ID NO:5 and degenerates thereof.
 29. An isolated nucleic acidsequence encoding a S. epidermidis protein selected from the groupconsisting of SdrG N1N2N3, SdrG N2N3 and SdrG TR2.
 30. The isolatednucleic acid sequence according to claim 29 having a sequence selectedfrom the group consisting of SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:5,and degenerates thereof.